Spatial mRNA / protein co-assay using aptamers

Abstract

The present disclosure generally relates to methods of making spatial proteome and / or transcriptome sequencing libraries. In some aspects, the spatial proteome and / or transcriptome sequencing library from a biological sample can be used to determine genetic maps and help diagnose a subject having or at risk of having a condition, and improve treatment of the subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 477,096, filed on December 23, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure generally relates to methods of preparing spatial proteomic sequencing libraries. Summary of the invention

[0004] Need for the technology that both spatial transcriptome and spatial proteome are mapped in the same tissue slice.Co-localization of protein and mRNA signal will make it better to understand how mRNA and protein expression are co-regulated.Some diseases (such as and not limited to Alzheimer's disease (Alzheimer's disease)) are characterized by abnormal protein deposition, and understanding how to change gene expression near these protein deposits can illustrate the potential mechanism of these diseases.The aptamer targeting cell membrane or nuclear membrane protein can also be used to limit cell boundary and nuclear boundary, thereby simplifying the cell segmentation of spatial transcriptome data.

[0005] Commercially available non-in situ spatial assays that can detect both proteins and mRNA are limited to using immunofluorescence to detect only a few proteins. Future products that can detect more proteins are antibodies based on oligomer conjugation. However, aptamers can also be used for in situ detection of proteins and have several advantages over antibodies. Aptamers are oligonucleotides (e.g., DNA or RNA oligonucleotides) that can specifically bind to proteins. Due to their smaller size compared to antibodies, aptamers can more easily diffuse into tissues. Aptamers are more stable and less sensitive to temperature and pH changes. Compared with antibodies, aptamers can also be manufactured more reproducibly and on a larger scale.

[0006] Aptamers can be modified with sequences to enable capture on barcoded surfaces. However, there are several challenges in designing spatial mRNA / protein co-assays:

[0007] 1. Adding long sequences to the aptamer to enable capture can affect the binding of the aptamer to the protein.

[0008] 2. Aptamers are difficult to remove from their target proteins.

[0009] 3. Free aptamers that are not bound to proteins must be prevented from binding to the barcoded surface.

[0010] 4. Proteins are much more abundant than mRNA molecules, and protein levels span a large dynamic range.

[0011] In various aspects, the present disclosure provides methods for using aptamers in spatial mRNA / protein co-assays that address these challenges.

[0012] In some aspects, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at a 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; (b) contacting a plurality of aptamers with the biological sample on the surface , the contacting causes a single aptamer in the plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer in the plurality of aptamers comprises (i) the target nucleotide sequence; (ii) an aptamer barcode nucleotide sequence; and (iii) a cleavage site; (c) removing aptamers in the plurality of aptamers that are not associated with proteins in the biological sample; (d) cleaving the plurality of aptamers to release (i) the target nucleotide sequence, and (ii) the aptamer barcode nucleotide sequence, thereby causing the target nucleotide sequence to associate with the capture nucleotide sequence, and thereby preparing the spatial proteome sequencing library. In some aspects, the UMI comprises a spatial barcode nucleotide sequence and is included in the aptamer instead of the capture nucleotide sequence. In some aspects, the surface further comprises a blocker nucleic acid that hybridizes to at least a portion of the capture nucleotide sequence. In some aspects, after step (c), the blocker nucleic acid is removed from the capture oligonucleotide. In various aspects, the plurality of aptamers are cleaved by ultraviolet radiation, enzymes, or chemical cleavage. In some aspects, the methods of the present disclosure further include (e) extending the capture nucleotide sequence to generate copies of the single aptamers, thereby generating an extended capture oligonucleotide. In some aspects, the methods of the present disclosure further include (f) adding a template switching oligonucleotide (TSO) to the 3' end of the extended capture oligonucleotide.

[0013] In some aspects, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at a 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; (b) contacting a plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer of the plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer of the plurality of aptamers comprises (i) the target nucleotide sequence; (ii) an aptamer barcode nucleotide sequence; and (iii) a truncated adapter nucleotide sequence; (c) removing aptamers of the plurality of aptamers that are not associated with proteins in the biological sample; and (d) eluting the single aptamers from the single proteins, thereby causing the target nucleotide sequence to associate with the capture nucleotide sequence, and thereby preparing the spatial proteome sequencing library. In some aspects, the surface also includes a sealant nucleic acid that hybridizes to at least a portion of the capture nucleotide sequence. In other aspects, after step (c), the sealant nucleic acid is removed from the capture oligonucleotide. In some aspects, the method of the present disclosure also includes (e) extending the capture nucleotide sequence to produce copies of these single aptamers, thereby producing extended capture oligonucleotides. In some aspects, the method of the present disclosure also includes (f) hybridizing and extending the truncated adapter nucleotide sequence with the full-length adapter nucleotide sequence primer to synthesize the second chain.

[0014] In another aspect, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each capture oligonucleotide of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at the 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; (b) contacting a plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer complex of the plurality of aptamer complexes to associate with a single protein in the biological sample, wherein the plurality of aptamer complexes Each aptamer complex of comprises: (1) an aptamer, the aptamer comprising (i) the capture nucleotide sequence; and (ii) an aptamer-specific nucleotide sequence; and (2) an oligonucleotide hybridized to the aptamer before the contacting, the oligonucleotide comprising (i) the target nucleotide sequence; (ii) a sequence complementary to the aptamer-specific nucleotide sequence; and (iii) an aptamer barcode nucleotide sequence, wherein after the association of a single aptamer complex in the plurality of aptamer complexes with a single protein in the biological sample, the oligonucleotide is released from the aptamer, thereby allowing the target nucleotide sequence of the released oligonucleotide to associate with the capture nucleotide sequence of the capture oligonucleotide in the plurality of capture oligonucleotides; thereby preparing the spatial proteome sequencing library. In some aspects, the length of the aptamer-specific nucleotide sequence is about 5 to about 20 nucleotides. In other aspects, the length of the aptamer-specific nucleotide sequence is about 10 nucleotides. In some aspects, the surface also includes a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence. In other aspects, after the contacting, the blocker nucleic acid is removed from the capture oligonucleotide. In some aspects, the association of a single aptamer complex in the multiple aptamer complexes with a single protein in the biological sample causes the oligonucleotide to be released from the aptamer. In some aspects, after the association of a single aptamer complex in the multiple aptamer complexes with a single protein in the biological sample, conditions are changed so that the oligonucleotide is released from the aptamer. In other aspects, the condition is temperature, pH or salt concentration. In still other aspects, after the association of a single aptamer complex in the multiple aptamer complexes with a single protein in the biological sample, formamide is added so that the oligonucleotide is released from the aptamer. In various aspects, the blocker oligonucleotide is removed from the capture oligonucleotide by exonuclease digestion. In other aspects, the exonuclease digestion is performed using T7 exonuclease or lambda exonuclease. In some aspects, these aptamers include a detectable portion. In some aspects, the detectable portion is a fluorescent portion.

[0015] In another aspect, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at the 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; (b) contacting a plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer of the plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer of the plurality of aptamers comprises (i) the target nucleotide sequence; and (ii) an aptamer barcode nucleotide sequence; (c) removing aptamers of the plurality of aptamers that are not associated with proteins in the biological sample; and (d) eluting the single aptamers from the single proteins, thereby causing the target nucleotide sequence to hybridize with the capture nucleotide sequence, and thereby preparing the spatial proteome sequencing library. In some aspects, the multiple capture oligonucleotides include a cleavable site at the 5' end. In some aspects, step (d) also includes contacting at least one of the multiple aptamers with a sealant nucleic acid, thereby forming a sealed aptamer, wherein the sealant nucleic acid is complementary to the target nucleotide sequence, and wherein the sealed aptamer cannot be associated with the capture nucleotide sequence. In some aspects, the surface also includes a sealant nucleic acid hybridized with at least a portion of the capture nucleotide sequence. In some aspects, after step (c), the sealant nucleic acid is removed from the capture oligonucleotide. In some aspects, the elution in step (d) includes digesting these proteins in the biological sample or competing with excess aptamers. In some aspects, the method also includes (e) extending the capture nucleotide sequence to produce copies of these single aptamers, thereby producing extended capture oligonucleotides. In some aspects, step (e) further comprises hybridizing a plurality of aptamer barcoded oligonucleotides to the extended capture oligonucleotides, and extending the extended capture oligonucleotides, thereby producing a plurality of barcoded capture oligonucleotides, wherein each of the aptamer barcoded oligonucleotides comprises at least a portion of a single aptamer sequence. In some aspects, the plurality of aptamer barcoded oligonucleotides comprises a plurality of aptamer blocker nucleic acids, wherein each of the aptamer blocker nucleic acids comprises at least a portion of a single aptamer sequence.

[0016] In some aspects, the method further comprises contacting a second plurality of aptamers with the biological sample on the surface, the contact causing a single aptamer in the second plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer in the second plurality of aptamers comprises a detectable portion. In some aspects, the detectable portion is a fluorophore. In some aspects, each aptamer in the second plurality of aptamers comprises the target nucleotide sequence and a truncated adapter nucleotide sequence. In some aspects, each aptamer in the second plurality of aptamers further comprises an aptamer barcode nucleotide sequence. In some aspects, after contacting the second plurality of aptamers with the biological sample on the surface, the method further comprises imaging the biological sample, thereby obtaining an image of the biological sample. In some aspects, the method does not include contacting the biological sample with a hematoxylin and eosin (H&E) staining reagent. In some aspects, at least one aptamer in the second plurality of aptamers is specific for cell membrane-associated proteins. In some aspects, at least one aptamer in the second plurality of aptamers is specific for nuclear membrane-associated proteins. In some aspects, at least one aptamer in the second plurality of aptamers is specific for a cell membrane-associated protein, and at least one aptamer in the second plurality of aptamers is specific for a nuclear membrane-associated protein, and the detectable portion included by the at least one aptamer specific for the nuclear membrane-associated protein is different from the detectable portion included by the at least one aptamer specific for the cell membrane-associated protein. In some aspects, the at least one aptamer specific for the nuclear membrane-associated protein includes an aptamer barcode nucleotide sequence that is different from the aptamer barcode nucleotide sequence included by the at least one aptamer specific for the cell membrane-associated protein. In some aspects, the cell membrane-associated protein is E-cadherin, N-cadherin, or Na + / K + -ATPase. In some aspects, the nuclear membrane-associated protein is a nuclear pore complex protein.

[0017] In any aspect or aspects of the present disclosure, the biological sample is from a mammal. In a further aspect, the biological sample is from a human.

[0018] In other aspects, the disclosure provides a method for identifying the disease in a subject suffering from the disease or at risk of suffering from the disease, the method comprising: i) generating a spatial proteome and / or transcriptome library from a biological sample from the subject according to the method disclosed herein, ii) comparing the proteome and / or genetic information from the sample proteome and / or transcriptome library with a control proteome and / or transcriptome library, iii) identifying the genetic variation associated with the disease in the sample proteome and / or transcriptome library. In some aspects, the disease is a neurodegenerative disease. In other aspects, the disease is Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is a spatial proteomic workflow using aptamers tagged with a polyA sequence at the 3' end and a truncated B15 adaptor at the 5' end.

[0020] Figure 2 It is shown how the methods provided herein can be modified to be compatible with spatial protein / mRNA co-assays.

[0021] Figure 3 An exemplary variation of the workflow of the present disclosure using an aptamer comprising a cleavable tag is shown.

[0022] Figure 4 An exemplary variation of the disclosed workflow using a tagged aptamer bound to single-stranded DNA is shown, where the single-stranded DNA is released from the tagged aptamer upon protein binding.

[0023] Figure 5 Depicted are exemplary methods provided by the present disclosure that address the problem of accommodating a large dynamic range and protein copy number.

[0024] Figure 6A-6F An exemplary variation of the disclosed workflow using modified aptamers comprising unique barcode and adapter sequences is shown, with a blocker oligonucleotide complementary to the aptamer adapter sequence included to accommodate a large dynamic range of protein abundance. Fig. 6A is a representative illustration of the cellular environment, including a mixture of proteins (labeled "1," "2," and "X" for mRNA-binding proteins) and mRNA. Figure 6B The step of permeabilizing the cells to allow the modified aptamer to enter the cells is shown. A washing step (not shown) removes unbound aptamer. Figure 6C Immobilized surface primers hybridized to blocking oligonucleotides are shown. These surface primers can have the same or different capture sequences. Fig.6D The step of deblocking the surface primer to expose the free 3' end is shown. Fig. 6E Shown are the steps of digesting the protein to release the mRNA and aptamer, and optionally adding a dynamic range compression (DRC) blocker oligonucleotide specific for the abundant protein aptamer. Fig. 6F The steps of capturing the released mRNA and aptamer barcode on an immobilized surface primer followed by reverse transcription and polymerase extension to produce covalently attached complementary sequences of the mRNA and aptamer barcode are shown. In some aspects, the mRNA is captured on the surface after permeabilization in the presence of the aptamer to minimize the loss of cellular mRNA during processing. In some aspects, the mRNA capture sequence is unblocked and the aptamer capture sequence is blocked.

[0025] Figure 7A-7DAn exemplary variation of the disclosed workflow using a DRC blocking agent to maintain abundant SOMA aggregates in solution is shown. Fig. 7A The released mRNA and SOMA polymers are shown to be copied on the immobilized surface primers by reverse transcription and polymerase extension after cell permeabilization, aptamer binding and protein digestion. STUBBY corresponds to the universal adapter sequence at the 3' end of the aptamer (i.e., SOMA polymer). DRC blocking agent (i.e., a blocking agent oligomer including the complement of the STUBBY sequence and the complement of the SOMA polymer sequence) is added to the SOMA polymer. Figure 7B The following steps are shown: After reverse transcription and polymerase extension, unbound mRNA and SOMA-mers are washed away, leaving behind immobilized, extended surface primers, including cDNA of the mRNA or the complement of the STUBBY and SOMA-mer sequences. Figure 7C The steps of adding a SOMAmer barcode to an extended surface primer comprising a complementary sequence to the SOMAmer sequence are shown, wherein an oligomer comprising the SOMAmer-SEQ sequence, the barcode sequence, and the B-15' sequencing primer sequence is hybridized to the extended surface primer and copied. A "dummy" oligomer is included that lacks the barcode sequence and the sequencing primer sequence and can act as a DRC blocker for the highly abundant SOMAmers. Fig.7D A cleavage step is shown to release the extended surface primer including the SOMAmer barcode and the B-15 adapter sequence, followed by a library preparation step including the addition of sequencing adapters using PCR. Sequencing the barcodes of SOMAmers provides an alternative way to identify SOMAmer sequences without sequencing them directly.

[0026] Figure 8A-8B An exemplary variation of the disclosed workflow using fluorescently labeled aptamers targeted to the cell membrane is shown, for example for cell segmentation analysis and image alignment processing. Fig. 8A The following steps are shown: adding a fluorescently labeled aptamer to the protein, allowing any unbound aptamer to flow away, and then imaging the tissue to visualize bound aptamer.The immobilized surface primer on the solid support is blocked with, for example, a complementary blocking oligonucleotide. Figure 8B The following steps are shown: removing the blocking oligomer from the surface primer using, for example, T7 or λ exonuclease; eluting the protein-bound aptamer with an excess of unlabeled aptamer; capturing the eluted aptamer; extending the surface primer to copy the aptamer to the surface; and performing second-strand synthesis using an intact B15 adapter sequence oligomer hybridized to a truncated B15 adapter.

[0027] Figure 9A-9B Various examples of cell membrane-targeting aptamers and nuclear membrane-targeting aptamers are shown. Fig. 9AVarious types of cell membrane targeting aptamers are shown that include a common fluorophore and barcode to, for example, enable membrane detection across different tissue types. Each aptamer can include a cell membrane barcode and a truncated B15 adaptor sequence. Fig. 9B Exemplary aptamers for co-detection of cell membrane and nuclear membrane proteins using differently labeled aptamers (eg, aptamers with different fluorophore and barcode sequences) are shown. DETAILED DESCRIPTION

[0028] The emerging field of spatial protein genomics is being driven by the development of new technologies that allow single cell groups to be mapped to their spatial positions in tissue slices. A method for spatial mapping of single cell transcriptomes involves the use of a surface coated with barcoded oligonucleotides, wherein the spatial position of each barcode is known. Barcoded oligonucleotides are positioned in a single feature, wherein each oligonucleotide in the same feature carries the same spatial barcode. Different embodiments of this surface include arrays of beads, spot arrays, aggregation flow cells or aggregated particles arranged on the surface. These oligonucleotides also include oligo (dT) capture sequences, which are combined with mRNA and serve as primers for reverse transcription. Then, tissue sections are placed on this surface, and polyA mRNA molecules in the tissue diffuse into the features and are captured on the surface. The captured RNA is reversely transcribed into cDNA, thereby connecting the spatial barcode to the cDNA sequence. Library preparation is then performed, and sequencing is performed on a standard (e.g., Illumina) sequencer. During analysis, spatial barcodes are used to map the physical position of the molecules from which the reads are derived.

[0029] the term

[0030] As used in this specification and the enumerated paragraphs herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0031] "About" and "approximately" generally refer to an acceptable degree of error for the amount measured given the nature or precision of the measurement. Exemplary degrees of error are within 20-25% of the value or range of values, such as within 20%, 10%, 5%, 4%, 3%, 2% or 1%.

[0032] As used herein, the terms "includes," "including," "includes," "including," "contains," "containing," "have," "having," and any variations thereof, are intended to cover a non-exclusive inclusion such that a process, method, product defined by a method, or composition of matter that includes, contains, or contains an element or list of elements includes not only those elements, but may include other elements not expressly listed or inherent to such process, method, product defined by a method, or composition of matter. Similarly, "includes," "including," "having," and "containing" are interchangeable and are not intended to be limiting.

[0033] As used herein, "surface" may refer to a portion of a substrate or support structure that can contact a reagent, bead, or analyte. The surface may be substantially flat or planar. Alternatively, the surface may be rounded or contoured. Exemplary contours that may be included on the surface are holes (e.g., micropores or nanopores), depressions, columns, ridges, channels, and the like. Exemplary materials that may be used as substrates or support structures include: glass, such as modified or functionalized glass; plastics, such as acrylic acid, polystyrene, or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane, or Teflon; polysaccharides or cross-linked polysaccharides, such as agarose or agarose gel; nylon; nitrocellulose; resins; silica or silica-based materials, including silicon and modified silicon, carbon fibers; metals; inorganic glass; fiber bundles, or a variety of other polymers. A single material or a mixture of several different materials may form a surface that can be used in certain examples. In some examples, the surface includes holes (e.g., micropores or nanopores). In some aspects, the surface comprises wells in an array of wells (e.g., microwells or nanowells) on a solid support of glass, silicon, plastic, or other suitable gel with patterned covalent attachments, such as poly(N-(5-azidoacetamidopentyl)acrylamide-coacrylamide) (PAZAM, see, e.g., U.S. Patent Application Publication No. 2014 / 0079923A1, which is incorporated herein by reference). In some examples, the support structure may include one or more layers. Non-limiting examples of surfaces include bead arrays, spotted arrays, aggregated particles arranged on a chip surface, membranes, multiwell plates, and flow cells.

[0034] In one aspect, "surface" and / or "substrate" disclosed herein may also include islands or clusters of immobilized capture agents or capture oligonucleotides. The island or cluster can be generated on the surface of a substrate (e.g., a flow cell) by using bridge amplification. In this case, the substrate includes a plurality of immobilized capture oligonucleotides on the surface of the substrate, which are combined with complementary adapter regions present in nearby primers or oligonucleotides to form a bridge-like structure; these bridge-like structures are then extended using a polymerase to produce double-stranded molecules, which are then denatured to leave single-stranded capture oligomers anchored to the substrate. After multiple iterations of the aforementioned process, an island or cluster of immobilized capture oligonucleotides is generated. Examples of the aforementioned processes that can be used with the methods and compositions disclosed herein can be found in WO 2022 / 015913 Al, which is incorporated herein by reference in its entirety. In a particular aspect, nearby primers or oligonucleotides are attached to a substrate (e.g., a flow cell) by a selectively cleavable joint. The shape of each island or cluster can be roughly circular or elliptical. Each island or cluster may have an average diameter of 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, 1050nm, 1100nm, 1200nm, or a range including any two of the foregoing diameters or between any two of the foregoing diameters. In another aspect, each 1 mm 2 The surface area of ​​the substrate (e.g., a circulation cell) includes 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1 million, 1.1 million, 1.2 million, 1.3 million, 1.4 million, 1.5 million, 1.6 million, 1.7 million, 1.8 million, 1.9 million, 2 million, 2.1 million, 2.2 million, 2.3 million, 2.4 million, or 2.5 million clusters, or includes any two of the foregoing numbers or a range between any two of the foregoing numbers. In certain aspects, as disclosed herein, a "substrate" includes islands or clusters of immobilized capture oligonucleotides, which include adapter sequences, spatial address sequences, optional sequence primer sites, and capture portions for targeted analytes. In yet another aspect, each cluster or island on a substrate (e.g., a circulation cell) includes a capture oligonucleotide having a unique spatial address sequence, so that the x, y position of each cluster or island can be identified. In this case, the x,y position of each cluster or island can be determined by decoding the spatial address sequence. The method of decoding the spatial address sequence includes but is not limited to the hybridization decoding or sequencing decoding method disclosed herein.

[0035] As used herein, the term "gap region" refers to an area in a substrate or on a surface that is separated from other areas of the substrate or surface. For example, a gap region may separate one feature of an array from another feature of the array. Two regions separated from each other may be discrete and lack contact with each other. In another example, a gap region may separate a first portion of a feature from a second portion of a feature. The separation provided by the gap region may be partial separation or complete separation. The gap region typically has a surface material different from the surface material of the features on the surface. For example, a feature of an array may have a capture agent or capture oligonucleotide in an amount or concentration that exceeds the amount or concentration present at the gap region. In some aspects, a capture agent or primer may not be present at the gap region.

[0036] In some aspects, the substrate includes an array of holes or depressions in the surface. This can be manufactured using a variety of techniques as is generally known in the art, including but not limited to photolithography, imprinting techniques, molding techniques, and micro-etching techniques. Those skilled in the art will appreciate that the technique used will depend on the composition and shape of the array substrate.

[0037] Exemplary flow cells include, but are not limited to, those used in nucleic acid sequencing devices, such as those used in the Genome or or for SOLiD commercialized by Life Technologies (Carlsbad, Calif.) TM or Ion Torrent TM The flow cell of sequencing platform.Exemplary flow cell and its manufacture and use method are also described in for example WO2014 / 142841 A1, U.S. Patent Application Publication No. 2010 / 0111768A1 and U.S. Patent No. 8,951,781, each of these patents is incorporated herein by reference.The flow cell can be a "non-patterned flow cell", wherein the surface of the flow cell includes the feature of random or semi-random arrangement (for example, including the cluster of oligonucleotide or the region of island).Alternatively, the flow cell can be a "patterned flow cell", wherein the flow cell includes the feature (for example, nanopore) at the fixed position on the surface across the flow cell.The feature of "patterned flow cell" can also include the cluster or island of immobilized oligonucleotide or immobilized oligonucleotide."patterned flow cell" can be an "ordered substrate", because the feature of the patterned flow cell has the x, y spatial address of distribution or the x, y spatial address that can be easily determined.

[0038] "Complementary" means that an oligonucleotide includes a nucleotide sequence that can form a double-stranded structure by matching base pairs with another oligonucleotide or a portion thereof. "Complementary" means that an oligonucleotide has at least 85%, 90%, 95%, 98%, 99% or 100% overall sequence identity with a complementary sequence.

[0039] In any aspect or aspects of the present disclosure, the methods described herein include sequencing procedures, such as and not limited to synthetic sequencing (SBS) technology or nanopore sequencing. In short, SBS can be initiated by contacting a barcode with one or more labeled nucleotides, DNA polymerases, etc. The features of primers extended using a sequence comprising a barcode as a template will incorporate detected labeled nucleotides. Optionally, the labeled nucleotides can also include a reversible termination property that terminates further primer extension once nucleotides are added to the primer. For example, a nucleotide analog with a reversible terminator portion can be added to the primer so that subsequent extension does not occur until a deblocking agent is delivered to remove the portion. Therefore, for aspects using reversible termination, deblocking agents can be delivered to a circulation pool (before or after detection occurs). Washing can be performed between each delivery step. Then, the cycle can be repeated n times, so that the primer extends n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluid systems, and detection platforms that can be readily adapted for use with libraries generated by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008); WO 04 / 018497; WO 91 / 06678; WO 07 / 123744; U.S. Pat. Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, or 7,405,281; and U.S. Patent Application Publication No. 2008 / 0108082A1, each of which is incorporated herein by reference.

[0040] As used herein, "primer" is a nucleic acid molecule that can hybridize with a target sequence, such as an adapter attached to a library fragment. In some aspects, an amplification primer can be used as a starting point for template amplification and cluster generation. For example, a synthetic nucleic acid (template) chain may include a site to which a primer (e.g., a sequencing primer) can hybridize, so as to initiate the synthesis of a new chain complementary to the synthetic nucleic acid chain. Any primer may include any combination of nucleotides or their analogs. In some examples, a primer is a single-stranded oligonucleotide or polynucleotide. The primer length can be any number of base lengths and can include a variety of non-natural nucleotides. In various aspects, a sequencing primer is a short chain, ranging from 5 to 60 bases, 10 to 60 bases, 10 to 20 bases, 10 to 30 bases, 10 to 40 bases, 10 to 50 bases, or 20 to 40 bases. Those skilled in the art can adjust these factors to provide optimal hybridization and signal generation for a given hybridization program. Primer allows to add nucleotide residues thereto under suitable conditions, or synthesize oligonucleotides or polynucleotides therefrom. On the one hand, primer is a DNA primer, i.e., a primer composed of deoxyribonucleotide residues or mainly composed of deoxyribonucleotide residues. Primer is designed to have a sequence of complementary sequence in the template / target DNA region hybridized by the primer. By forming a phosphodiester bond, nucleotide residues are added to the 3' end of the primer to produce DNA extension products. By forming a phosphodiester bond, nucleotide residues are added to the 3' end of the DNA extension product to produce other DNA extension products. On the other hand, primer is an RNA primer. In all aspects, primer hybridizes with target polynucleotides." Primer " is complementary to the polynucleotide template, and by hydrogen bonding or hybridization with the template, compound, to produce primer / template complex, to initiate polymerase synthesis, the primer is extended by adding the covalently bonded bases connected at its 3' end complementary to the template in the DNA synthesis process.

[0041] As used herein, the term "unique molecular identifier" or "UMI" refers to a random, non-random, or semi-random molecular tag that can be attached to a nucleic acid. When incorporated into a nucleic acid, the UMI can be used to correct for subsequent amplification bias by directly counting unique molecular identifiers (UMIs) sequenced after amplification. UMIs can be attached to similar nucleic acids (e.g., adapters) so that each nucleic acid is unique. In some aspects, the UMI comprises a spatial barcode.

[0042] As used herein, a "semi-random" nucleotide sequence comprises or consists of a partially predetermined nucleotide sequence combined with a random nucleotide sequence.

[0043] As used herein, the term "adapter" generally refers to any linear nucleic acid molecule that can be added to an oligonucleotide of the present disclosure. In some aspects, the adaptor is copied to a library molecule using a templated polymerase synthesis. In some aspects, the adaptor includes two reverse complementary oligonucleotides that form a double-stranded structure. In some aspects, the adaptor includes two oligonucleotides that are complementary at one part and mispaired at another part, thereby forming a Y-shaped or forked adaptor that is double-stranded at the complementary part and has two loose overhangs at the mispaired part. In some aspects, the adaptor is a template switching oligonucleotide (TSO) adaptor.

[0044] The term "template switching oligonucleotide" refers to an oligonucleotide template to which the polymerase activity is switched from an initial template (e.g., a single-stranded nucleic acid provided by a sample of the present invention). In one aspect of the present invention, the template switching oligonucleotide is a DNA / RNA hybrid oligonucleotide that is used by a template-dependent DNA or RNA polymerase (preferably RT, preferably MMLVRT) to continue reverse transcription after the enzyme (preferably MMLV RT) reaches the 5' end of the template nucleic acid and adds nucleotides to the 3' end of the synthesized cDNA or cRNA chain through its terminal transferase activity, i.e., template-independent. The 3' end of the TSO hybridizes with the nucleotides added by the terminal transferase activity of the template-dependent DNA or RNA polymerase, thereby effectively extending the 5' end of the template DNA or RNA, so that the template-dependent DNA or RNA polymerase (preferably RT, more preferably MMLV RT) also reversely transcribes the remaining 5' portion of the TSO, which includes the defined sequence to be added to the 5' end of the template nucleic acid. The TSO may include one or more modified or non-naturally occurring nucleotides (or their analogs). For example, the template-switching oligonucleotide can include one or more nucleotide analogs (e.g., LNA, FANA, 2'-O-methyl ribonucleotides, 2'-fluoro ribonucleotides, etc.), linkage modifications (e.g., phosphorothioate, 3'-3' and 5'-5' reverse linkages), 5' and / or 3' end modifications (e.g., 5' and / or 3' amino, biotin, DIG, phosphate, thiol, dye, quencher, etc.), one or more fluorescently labeled nucleotides, or any other feature that provides the desired function to the template-switching oligonucleotide.

[0045] When referring to the example of adapters, the terms "P5" and "P7" may be used. The terms "P5'" (P5 primer) and "P7'" (P7 primer) refer to the complementary sequences of P5 and P7, respectively. It will be understood that any suitable adapter can be used in the methods presented herein, and the use of P5 and P7 is merely exemplary. The use of adapters such as P5 and P7 or their complementary sequences on a circulation cell is known in the art, as exemplified by the disclosures of WO 2007 / 010251, WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941, WO 1998 / 044151, and WO 2000 / 018957, each of which is incorporated herein by reference in its entirety. For example, any suitable forward amplification primer, whether immobilized or in solution, can be used in the methods presented herein for hybridization with a complementary sequence and amplification of the sequence. Similarly, any suitable reverse amplification primer, whether immobilized or in solution, can be used in the methods presented herein for hybridization with complementary sequences and amplification of sequences. Those skilled in the art will understand how to design and use primer sequences suitable for capturing and / or amplifying nucleic acids presented herein.

[0046] As used herein, the term "barcode" is intended to mean a series of nucleotides in an oligonucleotide that can be used to identify an oligonucleotide, a spatial address on the surface (i.e., a "spatial barcode" or "spatial address sequence"), a characteristic of an oligonucleotide, and / or an operation that has been performed on an oligonucleotide. A barcode can be a naturally occurring nucleotide sequence or a nucleotide sequence that is not naturally present in an organism from which a barcoded nucleic acid is obtained. In various aspects, a barcode is unique in a pool of barcodes that are different from one another in sequence, or is uniquely associated with a specific sample polynucleotide in a sample polynucleotide pool. In various aspects, each barcode in an adapter pool is unique, so that a sequencing read including a barcode can be identified as being derived from a single sample polynucleotide molecule based solely on the barcode. In other aspects, a single barcode sequence can be used more than once, but adapters including repeated barcodes are associated with different sequences and / or different combinations of barcode adapters, so that sequence reads can still be uniquely distinguished as being derived from a single sample polynucleotide molecule based on barcode and adjacent sequence information (e.g., a sample polynucleotide sequence and / or one or more adjacent barcodes). In various aspects, the length of the barcode is about or at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75 or more nucleotides. In various aspects, the length of the barcode is shorter than 20, 15, 10, 9, 8, 7, 6 or 5 nucleotides. In various aspects, the length of the barcode is about 10 to about 50 nucleotides, such as a length of about 15 to about 40 or about 20 to about 30 nucleotides. In a pool of different barcodes, the barcodes can have the same or different lengths. Typically, the barcode has sufficient length and includes sufficiently different sequences to allow identification of sequencing reads derived from the same sample polynucleotide molecule. In various aspects, each barcode in the plurality of barcodes differs from each other barcode in the plurality of barcodes in at least three nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide positions. In some aspects, substantially degenerate barcodes can be referred to as random. In some aspects, a barcode can include a nucleic acid sequence from a pool of known sequences. In some aspects, a barcode can be predefined.

[0047] As used herein, "biological sample" may include one or more biological or chemical substances, such as nucleic acids, oligonucleotides, proteins, cells, tissues, organisms and / or biologically active chemical compounds, such as analogs or analogs of the aforementioned classes. In some cases, biological samples may include whole blood, lymph, serum, plasma, sweat, tears, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal secretions, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural effusion, transudate, exudate, gallbladder fluid, bile, urine, gastric juice, intestinal fluid, stool samples, liquid containing single or multiple cells, liquid containing organelles, fluidized tissue, fluidized organisms, viruses (including viral pathogens), liquid containing multicellular organisms, biological swabs and biological washes. In other examples, the sample can be derived from an organ, including, for example, an organ of the musculoskeletal system, such as a muscle, bone, tendon, or ligament; an organ of the digestive system, such as a salivary gland, pharynx, esophagus, stomach, small intestine, large intestine, liver, gall bladder, or pancreas; an organ of the respiratory system, such as the larynx, trachea, bronchi, lungs, or diaphragm; an organ of the urinary system, such as the kidney, ureter, bladder, or urethra; a reproductive organ, such as an ovary, fallopian tube, uterus, vagina, placenta, testis, epididymis, vas deferens, seminal vesicle, prostate, penis, or scrotum; an organ of the endocrine system, such as the pituitary gland, pineal gland, thyroid gland, parathyroid gland, or adrenal gland; an organ of the circulatory system, such as the heart, arteries, veins, or capillaries; an organ of the lymphatic system, such as a lymphatic vessel, lymph node, bone marrow, thymus, or spleen; an organ of the central nervous system, such as the brain, brainstem, cerebellum, spinal cord, cranial nerves, or spinal nerves; a sensory organ, such as an eye, ear, nose, or tongue; or an integumentary organ, such as the skin, subcutaneous tissue, or breast. In various aspects, tissue can be derived from multicellular organisms. In some aspects, tissue sections can be in contact with a surface, such as by placing the tissue on the surface. Tissue can be freshly excised from an organism, or it can be previously preserved, such as by freezing (e.g., fresh frozen tissue), being embedded in a material such as paraffin (e.g., formalin-fixed paraffin-embedded (FFPE) sample), formalin fixation, infiltration, dehydration, etc. Optionally, tissue sections can be attached to a surface, such as using the technology and composition described in, for example, U.S. Patent No. 11,390,912, which is incorporated herein by reference in its entirety. In some aspects, when tissue is in contact with a surface, tissue can be permeabilized and the cell lysis of the tissue. Any of a variety of processes can be used, such as those described above about lysed cells. Target protein and / or nucleic acid released from permeabilized tissue can be captured by capture oligonucleotides on the surface. The thickness of the tissue sample or other biological sample contacted with the surface in the method described herein can be any suitable thickness required. In representative aspects, the thickness will be at least 0.1 μm, 0.25 μm, 0.5 μm, 0.75 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, or more.Alternatively or additionally, the thickness of the biological sample in contact with the surface will be no more than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 0.25 μm, 0.1 μm or thinner.

[0048] As used herein, the term "permeable" refers to the property of a substance that allows certain materials to pass through a substance. "Permeable" can be used to describe a biological sample, such as a cell or a cell nucleus, in which an analyte in the biological sample can leave the biological sample. "Permeabilization" is the action taken to cause, for example, a biological sample (e.g., a cell) to release its analyte. In some examples, the permeabilization of a biological sample is accomplished by affecting the integrity (e.g., damage) of a biological sample membrane (e.g., a cell or nuclear membrane), such as by applying a protease or other enzyme that can interfere with the membrane, thereby allowing the analyte to diffuse outside the biological sample. In some aspects, the permeabilized biological sample does not release the biomolecules (e.g., proteins and / or nucleic acids) included in the sample.

[0049] As used herein, a "capture oligonucleotide" is generally an oligonucleotide that includes a nucleotide sequence that can hybridize or otherwise associate with an aptamer or other oligonucleotide as described herein (e.g., mRNA, a single-stranded oligonucleotide released from an aptamer complex after association of an aptamer complex with a protein, a probe that binds to an mRNA target). The nucleotide sequence that can hybridize or otherwise associate with an aptamer or other oligonucleotide is, for example, and not limited to, a universal sequence (e.g., a polyT sequence) or a target-specific sequence. The capture oligonucleotide may include additional elements, including, but not limited to, a unique molecular identifier (UMI), a spatial barcode, a primer sequence amplified therefrom (e.g., A14-ME), a sequence for generating a barcode feature (e.g., a P7 sequence for clustering and an SBS12 sequence used as a sequencing primer binding site), or a combination thereof.

[0050] As used herein, "universal sequence" refers to a common nucleotide sequence in a variety of capture oligonucleotides. The common nucleotide sequence can be, for example, a sequence complementary to the same adapter sequence. Universal capture oligonucleotides are suitable for probing a variety of different oligonucleotides without having to distinguish between different species, while target-specific capture sequences are suitable for distinguishing between different species. A non-limiting example of a universal sequence is a polyT nucleotide sequence.

[0051] As used herein, "hybridization" is intended to mean the non-covalent association of a first oligonucleotide with a second oligonucleotide along the length of those polymers to form a double-stranded "duplex". For example, two DNA oligonucleotide chains can associate by complementary base pairing. The strength of the association between the first oligonucleotide and the second oligonucleotide increases with the complementarity between the nucleotide sequences within those oligonucleotides. The strength of the hybridization between oligonucleotides can be measured by the melting temperature (T m) to characterize, at this melting temperature, 50% of the duplexes have oligonucleotide chains that dissociate from each other. Oligonucleotides that "partially" hybridize to each other mean that they have sequences that are complementary to each other, but such sequences only hybridize to each other along a portion of their length to form a partial duplex. Oligonucleotides that "cannot" hybridize include those oligonucleotides that are physically separated from each other, so that the number of their bases is not enough to contact each other in a way that hybridizes to each other. For example, hybridization can be carried out in a temperature range of 15 ° C to 95 ° C. In some aspects, hybridization is carried out at the following temperatures: about 20 ° C, about 25 ° C, about 30 ° C, about 35 ° C, about 40 ° C, about 45 ° C, about 50 ° C, about 55 ° C, about 60 ° C, about 65 ° C, about 70 ° C, about 75 ° C, about 80 ° C, about 85 ° C, about 90 ° C or about 95 ° C. In other aspects, the stringency of hybridization can be further changed by adding or removing the components of the buffer solution.

[0052] As used herein, the term "multiple numbers" is intended to mean a colony of two or more members, and these two or more members can be all the same, or two or more members can be different.Multiple numbers can be in the range of small, medium, large to extremely large sizes.The size of small multiple numbers can be in the range of, for example, from several members to tens of members.Multiple numbers of medium size can be in the range of, for example, tens of members to about 100 members or hundreds of members.Large multiple numbers can be in the range of, for example, about hundreds of members to about 1000 members, to thousands of members, and up to tens of thousands of members.Extremely large multiple numbers can be in the range of, for example, tens of thousands of members to about hundreds of thousands, one million, millions, tens of millions, and up to or exceeding hundreds of millions of members.Therefore, multiple numbers can be in the range of two to far more than 100 million members' size and as measured by the number of members' all sizes, between the above exemplary ranges and exceed the above exemplary ranges.Therefore, the definition of term is intended to include all integer values ​​greater than two.The upper limit of multiple numbers can be set, for example, by the theoretical limitation of oligonucleotides (for example, capture oligonucleotides) on the surface.

[0053] In some respects, nucleic acid includes a label. As used herein, the term "label (label or labels)" is used according to its simple and common meaning, and refers to a molecule that can directly or indirectly produce or cause a detectable signal by itself or with another molecule interaction. Non-limiting examples of detectable labels include fluorescent dyes, biotin, digoxin, haptens and epitopes. Generally, dyes are molecules, compounds or substances that can provide optically detectable signals, such as colorimetric, luminescent, bioluminescent, chemiluminescent, phosphorescent or fluorescent signals. In various aspects, a label is a dye. In various aspects, a dye is a fluorescent dye. Non-limiting examples of dyes, some of which are commercially available, include CF dyes (Biotium, Inc.), Alexa Fluor dyes (ThermoFisher), DyLight dyes (ThermoFisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes (Anaspec, Inc.). In various aspects, a particular nucleotide type is associated with a particular label such that identification of the label identifies the nucleotide associated therewith. In various aspects, the label is a luciferin that reacts with a luciferase to produce a detectable signal in response to one or more bases being incorporated into an extended complementary strand, such as in pyrophosphate sequencing. In various aspects, the nucleotide comprises a label (such as a dye). In various aspects, the label is not associated with any particular nucleotide, but detection of the label identifies whether one or more nucleotides with a known identity were added during the extension step (such as in the case of pyrophosphate sequencing). Examples of detectable agents (i.e., labels) include imaging agents, including fluorescent and luminescent substances, molecules or compositions, including but not limited to various organic or inorganic small molecules commonly referred to as "dyes", "labels" or "indicators". Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes and cyanine dyes. In various aspects, the detectable portion is a fluorescent molecule (e.g., an acridine dye, a cyanine dye, a fluoro dye, an oxazine dye, a phenanthridine dye or a rhodamine dye). In various aspects, the detectable portion is a fluorescent molecule (e.g., an acridine dye, a cyanine dye, a fluoro dye, an oxazine dye, a phenanthridine dye or a rhodamine dye). As described herein, the term "cyanine" or "cyanine moiety" refers to a detectable portion comprising two nitrogen groups separated by a polymethine chain. In various aspects, the cyanine moiety has three methine structures (i.e., cyanine 3 or Cy3). In various aspects, the cyanine moiety has five methine structures (i.e., cyanine 5 or Cy5). In various aspects, the cyanine moiety has 7 methine structures (ie, cyanine 7 or Cy7).

[0054] Oligonucleotides

[0055] Oligonucleotide is a polymer comprising nucleotides. Oligonucleotides of the present disclosure (e.g., aptamers) can have any length, and in various aspects, include DNA oligonucleotides, RNA oligonucleotides, analogs thereof, or combinations thereof. In any aspect or aspects described herein, oligonucleotides are single-stranded, double-stranded, or partially double-stranded.

[0056] Nucleotides may include naturally occurring nucleotides and functional analogs thereof. Examples of functional analogs are those that can hybridize with nucleic acids in a sequence-specific manner or can be used as templates for replicating specific nucleotide sequences. Naturally occurring nucleotides typically have a backbone containing a phosphodiester bond. Similar structures may have alternative backbone bonds, including any of a variety of backbone bonds known in the art. Naturally occurring nucleotides typically have deoxyribose (e.g., present in DNA) or ribose (e.g., present in RNA). Similar structures may have alternative sugar moieties, including any of a variety of sugar moieties known in the art. Nucleotides may include natural or non-natural bases. Natural DNA may include one or more of adenine, thymine, cytosine and / or guanine, and natural RNA may include one or more of adenine, uracil, cytosine and / or guanine. Any non-natural bases may be used, such as locked nucleic acids (LNA) and bridge nucleic acids (BNA). Exemplary modified nucleotides include inosine, xathanine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, The nucleotide analogs of the present invention can be substituted with 5- pyrimidine, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-thiol adenine or guanine, 8-sulfanyl adenine or guanine, 8-hydroxy adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, etc. As known in the art, some nucleotide analogs cannot become incorporated into the polynucleotide, for example nucleotide analogs, such as 5'-phosphosulfate adenosine. Nucleotide can contain any suitable number of phosphates, for example three, four, five, six or more than six phosphates.

[0057] Oligonucleotides contemplated by the present disclosure also include those with at least one modified internucleotide bond. In some aspects, the oligonucleotide is a peptide nucleic acid in whole or in part. The internucleoside bond of other modifications includes at least one phosphorothioate bond. The oligonucleotides of other modifications include those comprising one or more universal bases. "Universal base" refers to a molecule that can replace any one of the combinations of A, C, G, T and U in nucleic acid by forming a hydrogen bond without significant structural destabilization. Examples of universal bases include but are not limited to 5'-nitroindole-2'-deoxynucleoside, 3-nitropyrrole, inosine and hypoxanthine.

[0058] In various aspects, the length of the oligonucleotides of the present disclosure or their modified forms is generally from about 5 nucleotides to about 150 nucleotides. In other aspects, the length of the oligonucleotides of the present disclosure is from about 5 to about 125 nucleotides, from about 5 to about 100 nucleotides, from about 5 to about 90 nucleotides, from about 5 to about 50 nucleotides, from about 5 to about 45 nucleotides, from about 5 to about 40 nucleotides, from about 5 to about 35 nucleotides, from about 5 to about 30 nucleotides, from about 5 to about 25 nucleotides, from about 5 to about 20 nucleotides, from about 5 to about 15 nucleotides, from about 5 to about 10 nucleotides, from about 10 to about 150 nucleosides in length. Acid, about 10 to about 125 nucleotides in length, about 10 to about 100 nucleotides in length, about 10 to about 90, about 10 to about 50 nucleotides in length, about 10 to about 45 nucleotides in length, about 10 to about 40 nucleotides in length, about 10 to about 35 nucleotides in length, about 10 to about 30 nucleotides in length, about 10 to about 25 nucleotides in length, about 10 to about 20 nucleotides in length, about 10 to about 15 nucleotides in length, and all oligonucleotides of intermediate size lengths are specifically disclosed to the extent that the oligonucleotide is able to achieve the desired result.Thus, in various aspects, the oligonucleotides of the present disclosure are or are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 1 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 1In other aspects, the oligonucleotides of the present disclosure are no less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198

[0059] As used herein, when used to refer to a nucleic acid sequence, the term "poly T" or "poly A" is intended to mean a series of two or more thiamine (T) or adenine (A) bases, respectively. Poly T or poly A can include at least about 2, 5, 8, 10, 12, 15, 18, 20 or more T or A bases, respectively. Alternatively or additionally, poly T or poly A can include up to about 30, 20, 18, 15, 12, 10, 8, 5 or 2 T or A bases, respectively. In some aspects, the disclosure contemplates the use of a "polyTVN" sequence, which is a poly T sequence followed by V (any base except T) and N. In some aspects, the polyTVN sequence is used to bias reverse transcription toward the bases of the poly A tail on the mRNA molecule.

[0060] As used herein, when using with reference to oligonucleotides, the term "fixed" is intended to mean directly or indirectly attached to a substrate by covalent or non-covalent bonds. In some aspects, covalent attachment can be used, but all that is required is that the oligonucleotides remain fixed or attached to a surface under conditions intended to use a surface (e.g., in applications requiring nucleic acid capture, amplification and / or sequencing). The oligonucleotide to be used as a capture oligonucleotide can be fixed so that the 3' end can be used for enzymatic extension, and at least a portion of the sequence can hybridize with a complementary sequence. Fixation can occur by hybridization of oligonucleotides attached to the surface, in which case the fixed oligonucleotide or polynucleotide can be in a 3'-5' orientation. Alternatively, the fixation of oligonucleotides can include the use of a selectively cleavable joint. Examples of selectively cleavable joints include, but are not limited to, biotin-based molecules (e.g., desulphobiotin molecules (ddBio)), PC joints and recognition sites for rare cutting enzymes. Typically, selectively cleavable joints can be cleaved by heating, competitive binding, pH changes, chemical cleavage, enzymatic cleavage and / or photocleavage. Cleavage of the selectively cleavable linker results in release of the nucleic acid or portion thereof from the substrate or feature of the substrate.

[0061] Some aspects utilize inert substrates or matrix (e.g., slides, polymer beads, etc.), which have been functionalized, for example, by applying an intermediate material layer or coating comprising reactive groups, which allow covalent attachment to biomolecules, such as polynucleotides. Examples of such substrates include, but are not limited to, polyacrylamide hydrogels loaded on inert substrates (such as glass), especially polyacrylamide hydrogels as described in WO 2005 / 065814 and US2008 / 0280773, the contents of which are incorporated herein by reference in their entirety. In such aspects, biomolecules (e.g., polynucleotides) can be directly covalently attached to intermediate materials (e.g., hydrogels), but intermediate materials themselves can be non-covalently attached to substrates or matrix (e.g., glass substrates). The term "covalently attached to substrates" should be interpreted accordingly as encompassing this type of arrangement.

[0062] Exemplary covalent linkages include, for example, those produced using click chemistry techniques. Exemplary non-covalent linkages include, but are not limited to, nonspecific interactions (e.g., hydrogen bonding, ionic bonding, van der Waals interactions, etc.) or specific interactions (e.g., affinity interactions, receptor-ligand interactions, antibody epitope interactions, avidin-biotin interactions, streptavidin-biotin interactions, lectin-carbohydrate interactions, etc.). Exemplary linkages are described in U.S. Patents Nos. 6,737,236, 7,259,258, 7,375,234, and 7,427,678; and U.S. Patent Publication No. 2011 / 0059865A1, each of which is incorporated herein by reference.

[0063] As used herein, when used to refer to nucleic acids, the term "extension" is intended to mean the addition of at least one nucleotide or oligonucleotide to a nucleic acid. In certain aspects, one or more nucleotides can be added to the 3' end of a nucleic acid, for example, by polymerase catalysis (e.g., DNA polymerase, RNA polymerase, or reverse transcriptase). Chemical or enzymatic methods can be used to add one or more nucleotides to the 3' or 5' end of a nucleic acid. One or more oligonucleotides can be added to the 3' or 5' end of a nucleic acid, for example, by chemical or enzymatic (e.g., ligase catalysis) methods. Nucleic acids can be extended in a template-guided manner, whereby the extension product is complementary to the template nucleic acid, and the template nucleic acid hybridizes with the extended nucleic acid.

[0064] As used herein, the terms "DNA polymerase" and "nucleic acid polymerase" are used according to their general common meanings and refer to enzymes capable of synthesizing nucleic acid molecules from nucleotides (e.g., deoxyribonucleotides). Typically, a DNA polymerase adds nucleotides to the 3' end of a DNA chain, one nucleotide at a time. In various aspects, the DNA polymerase is a Pol I DNA polymerase, a Pol II DNA polymerase, a Pol III DNA polymerase, a Pol IV DNA polymerase, a Pol V DNA polymerase, a Pol β DNA polymerase, a Pol μ DNA polymerase, a Pol λ DNA polymerase, a Pol σ DNA polymerase, a Pol α DNA polymerase, a Pol δ DNA polymerase, a Pol ε DNA polymerase, a Pol η DNA polymerase, a Pol ι DNA polymerase, a Pol κ DNA polymerase, a Pol ζ DNA polymerase, a Pol γ DNA polymerase, a Pol θ DNA polymerase, a Pol υ DNA polymerase, or a thermophilic nucleic acid polymerase (e.g., Therminator γ, 9°N polymerase (exo), Therminator II, Therminator III, or Therminator IX). In various aspects, the DNA polymerase is a modified archaeal DNA polymerase. In all aspects, the polymerase is a reverse transcriptase. For example, the polymerase catalyzes the addition of the next correct nucleotide to the 3'-OH group of the primer through a phosphodiester bond, thereby chemically incorporating the nucleotide into the primer. Optionally, the polymerase used in the provided method is a progressive polymerase. Optionally, the polymerase used in the provided method is a distributed polymerase.

[0065] As used herein, the term "exonuclease activity" is used according to its common meaning in the art, and refers to the removal of nucleotides from nucleic acids by DNA polymerase. For example, during polymerization, nucleotides are added to the 3' end of the primer chain. Sometimes, DNA polymerase incorporates incorrect nucleotides into the 3'-OH end of the primer chain, wherein the incorrect nucleotides cannot form hydrogen bonds with the corresponding bases in the template chain. Due to the 3' to 5' exonuclease activity of DNA polymerase, such erroneously added nucleotides are removed from the primer. In various aspects, exonuclease activity can be referred to as "proofreading". When referring to 3'-5' exonuclease activity, it is understood that DNA polymerase promotes a hydrolysis reaction that destroys the phosphodiester bond at the 3' end of the polynucleotide chain to remove nucleotides. In various aspects, 3'-5' exonuclease activity refers to the continuous removal of nucleotides in single-stranded DNA in the 3'→5' direction, thereby releasing deoxyribonucleoside 5'-monophosphates one by one. Methods for quantifying exonuclease activity are known in the art, see, for example, Southworth et al., Proceedings of the National Academy of Sciences (PNAS) Vol. 93, 8281-8285 (1996). In various aspects, 5'-3' exonuclease activity refers to the continuous removal of nucleotides in double-stranded DNA in the 5'→3' direction. In various aspects, the 5'-3' exonuclease is a lambda exonuclease. For example, a lambda exonuclease catalyzes the removal of 5' single nucleotides from a duplex DNA (preferably a 5' phosphorylated double-stranded DNA). In other aspects, the 5'-3' exonuclease is Escherichia coli (E. coli) DNA polymerase I.

[0066] As used herein, the term "cleavable linker" or "cleavable part" refers to a divalent or monovalent part that can be separated (e.g., detached, split, unlinked, hydrolyzed, and the stable bond in the part is broken) into a unique entity. The cleavable linker is cleavable (e.g., specifically cleavable) in response to external stimuli (e.g., enzymes, nucleophilic / alkaline reagents, reducing agents, photoirradiation, electrophilic / acidic reagents, organic metals and metal reagents or oxidants). A chemically cleavable linker refers to a linker that can be split in response to the presence of a chemical substance (e.g., acid, alkali, oxidant, reducing agent, Pd (0), tris-(2-carboxyethyl) phosphine, dilute nitrous acid, fluoride, tris (3-hydroxypropyl) phosphine), sodium dithionite (Na2S2O4) or hydrazine (N2H4)). A chemically cleavable linker is non-enzymatically cleavable. In various aspects, the cleavable linker is cleaved by contacting the cleavable linker with a cleaving agent. In various aspects, the cracking agent is a phosphine-containing reagent (for example, TCEP or THPP), sodium dithionite (Na2S2O4), a weak acid, hydrazine (N2H4), Pd (O) or light irradiation (for example, ultraviolet irradiation). In various aspects, cracking includes removal. In the context of polynucleotides, "cleavable site" or "scissor-type bond" is a site that allows the control of cleavage polynucleotide chains (for example, joints, primers or polynucleotides) by chemical, enzymatic or photochemical means known in the art and described herein. The scissor-type site can refer to the key of the nucleotide between two other nucleotides in the nucleotide chain (that is, internucleoside bond). In various aspects, the scissor-type bond can be located at any position in one or more nucleic acid molecules, including at or near the end (for example, the 3' end of an oligonucleotide), or in the internal position of the one or more nucleic acid molecules. In various aspects, the conditions suitable for separating the scissor-type bond include regulating pH and / or temperature. In various aspects, the scissor-type site can include at least one acid-labile bond. For example, an acid-labile bond can include an aminophosphorothioate bond. In various aspects, the phosphoramidate bond can be hydrolyzed under acidic conditions, and these acidic conditions include weakly acidic conditions, such as trifluoroacetic acid and suitable temperature (for example, 30 ° C), or other conditions known in the art, such as Matthias Mag et al. "Tetrahedron Letters", Vol. 33, No. 48, 1992, 7319-7322. In various aspects, the scissor site can include at least one light-labile internucleoside bond (for example, o-nitrobenzyl bond, such as Walker et al., "Journal of the American Chemical Society (J.Am.Chem.Soc.)" 1988, 110, 21, described in 7170-7177), such as o-nitrobenzyloxymethyl or p-nitrobenzyloxymethyl. In various aspects, the scissor site includes at least one uracil nucleobase. In various aspects, uracil DNA glycosylase (UDG) or formamidopyrimidine DNA glycosylase Fpg can be used to crack uracil nucleobase.In various aspects, the scissor-type bond site includes a sequence-specific nicking site having a nucleotide sequence that is identified and nicked by a nicking endonuclease or uracil DNA glycosylase. In various aspects, the cleavable site can be cleaved by an enzyme or chemical reagent at or near a modified nucleotide or bond, which are collectively referred to as "cleaving agents" herein and in the claims. The example of a cleaving agent includes a DNA repair enzyme, a glycosylase, a DNA cleaving endonuclease or a ribonuclease. For example, uracil DNA glycosylase and endonuclease VIII (USER) can be used for the cleavage at a dUTP site. TM , NEB, Ipswich, Mass.) is implemented as described in U.S. Patent No. 7,435,572. In various aspects, when the modified nucleotide is a ribonucleotide, the cleavable site can be cleaved with an endoribonuclease. In various aspects, the cleavage extension product includes contacting the cleavable site with a cleavage agent, wherein the cleavage agent includes a reducing agent, sodium periodate, an RNA enzyme, a formamido pyrimidine DNA glycosylase (Fpg), an endonuclease, a restriction enzyme, or a uracil DNA glycosylase (UDG). In various aspects, the cleavage agent is an endonuclease, such as nuclease P1, AP endonuclease, T7 endonuclease, T4 endonuclease IV, Bal 31 endonuclease, endonuclease I (endo I), micrococcal nuclease, endonuclease II (endo VI, exo III), nuclease BAL-31 or mung bean nuclease. In various aspects, the cleavage agent includes a restriction endonuclease, including, for example, a IIS type restriction endonuclease. In various aspects, the cleavage agent is an exonuclease (e.g., RecBCD), a restriction nuclease, an endoribonuclease, an exoribonuclease, or an RNA enzyme (e.g., RNA enzyme I, II, or III). In various aspects, the cleavage agent is a restriction enzyme. In various aspects, the cleavage agent includes a glycosylase and one or more suitable endonucleases. In various aspects, the cleavage agent is performed under alkaline (e.g., pH greater than 8) buffer conditions between 40°C and 80°C (e.g., 65°C).

[0067] Aptamer

[0068] The methods of the present disclosure include the use of aptamers, which are oligonucleotides that can specifically bind to proteins. Generally, the present disclosure provides methods in which aptamers specifically bind to proteins in biological samples. After the aptamer binds to the protein, the aptamer (or a portion thereof) or the oligonucleotide hybridized to the aptamer is released from the protein and captured on the surface by the capture oligonucleotide. The aptamer sequence can then be copied, amplified, and sequenced to determine (i) the identity of the aptamer and therefore the identity of the protein, and (ii) the spatial information of the protein in the biological sample.

[0069] As used herein, "aptamers" and "SOMA polymers" are used interchangeably to refer to non-naturally occurring nucleic acids that have a desired effect on a target molecule. The desired effect includes, but is not limited to, binding to a target, catalytically changing a target, reacting with a target in a manner that modifies or changes a target or a functional activity of a target, covalently attaching to a target, and promoting a reaction between a target and another molecule. In one aspect, the effect is a specific binding affinity to a target molecule, such a target molecule being a three-dimensional chemical structure other than a polynucleotide that binds to a nucleic acid ligand by a mechanism that is independent of Watson / Crick base pairing or triple helix formation, wherein an aptamer is not a nucleic acid with a known physiological function that is bound by the target molecule. Aptamers for a given target include nucleic acids identified from a candidate mixture of nucleic acids by a method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids with increased affinity for the target relative to other nucleic acids in the candidate mixture can be partitioned from the remainder of the candidate mixture; (b) partitioning the nucleic acids with increased affinity from the remainder of the candidate mixture; and (c) amplifying the nucleic acids with increased affinity to produce a ligand-enriched nucleic acid mixture, thereby identifying aptamers for the target molecule. It is recognized that affinity interactions are a matter of degree; however, in this context, the "specific binding affinity" of an aptamer to its target means that the binding of the aptamer to its target has a much higher degree of affinity than its binding to other non-target components in the mixture or sample. An aptamer can include any suitable number of nucleotides. "Appatamer" refers to more than one set of such molecules. Different aptamers can have the same or different numbers of nucleotides. Aptamers can be DNA or RNA, and can be single-stranded, double-stranded, or include double-stranded or triple-stranded regions. Aptamers can be designed with any combination of desired base-modified nucleotides.

[0070] As used herein, "SOMA polymers" or slow off-rate modified aptamers refer to aptamers (including aptamers comprising at least one nucleotide with a hydrophobic modification) with an off-rate (t1 / 2) of ≥30 minutes. In some aspects, SOMA polymers are generated using the improved SELEX method described in U.S. Pat. No. 7,947,447, entitled "Method for Generating Aptamers with Improved Off-Rates", which is incorporated herein by reference.

[0071] Aptamers can be identified using any known method, including the SELEX method. See, for example, U.S. Pat. No. 5,475,096, entitled "Nucleic Acid Ligands." Once identified, aptamers can be prepared or synthesized according to any known method, including chemical synthesis methods and enzymatic synthesis methods.

[0072] As used herein, the term "aptamer-target affinity complex", "aptamer affinity complex" or "aptamer complex" refers to a non-covalent complex formed by the interaction of an aptamer with its target molecule. "Appamer-target affinity complex", "aptamer affinity complex" or "aptamer complex" refers to more than one set of such complexes. The aptamer-target affinity complex, aptamer affinity complex or aptamer complex can generally be reversible or dissociated by changes in environmental conditions (e.g., an increase in temperature, an increase in salt concentration, or the addition of a denaturant).

[0073] In some aspects, a non-covalent complex of an aptamer and its target is provided, wherein the aptamer has a Kd for the target of about 100 nM or less, wherein the dissociation rate of the aptamer from the target (as given by the half-life of the complex; t1 / 2) is greater than or equal to about 30 minutes; and / or wherein one, several or all pyrimidines in the nucleic acid sequence of the aptamer are modified at the 5' position of the base.

[0074] As used herein, "non-specific complex" refers to a non-covalent association between two or more molecules other than an aptamer and its target molecule. Because non-specific complexes are not selected based on affinity interactions between their constituent molecules, but rather represent interactions between molecular classes, the molecules associated in non-specific complexes will exhibit on average much lower mutual affinity than an aptamer and its target molecule, and will have a correspondingly higher dissociation rate. Non-specific complexes include complexes formed between an aptamer and a non-target molecule, an aptamer and another aptamer, a competitor and a non-target molecule, a competitor and a target molecule, an aptamer and a competitor, and a target molecule and a non-target molecule, as well as higher-order aggregates of aptamers, target molecules, non-target molecules, surfaces, and competitors.

[0075] As used herein, "target molecule", "analyte" and "target" are used interchangeably to refer to any molecule of interest to which an aptamer can bind with high affinity and specificity and can be present in a test sample. "Molecule of interest" includes any minor changes in a particular molecule, such as in the case of a protein, such as a minor change in the amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component that does not substantially change the identity of the molecule. Exemplary target molecules include proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affibodies, antibody mimetics, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, and any fragment or portion of any of the foregoing. An aptamer can be identified as having almost any chemical or biological molecule of any size, and therefore having almost any chemical or biological molecule of any size can be a suitable target. The target can also be modified to enhance the likelihood or intensity of interaction between the target and the aptamer. The target may also be modified to include a tag as defined above. In exemplary aspects, the target molecule is a protein. See U.S. Pat. No. 6,376,190, entitled "Modified SELEX Processes Without Purified Protein," in which the SELEX target is a peptide.

[0076] In various aspects, the aptamer of the present disclosure includes one or more elements, including but not limited to protein binding nucleotide sequences, complementary target nucleotide sequences, adapter nucleotide sequences or parts thereof, aptamer barcode nucleotide sequences, cleavage sites, aptamer-specific nucleotide sequences or combinations thereof. In some aspects, the aptamer of the present disclosure includes a capture nucleotide sequence, and the oligonucleotide hybridized with the aptamer includes a target nucleotide sequence that is at least partially complementary. As used herein, a "capture nucleotide sequence" is a sequence that can hybridize with a target nucleotide sequence on an aptamer or with an oligonucleotide hybridized with an aptamer. In some aspects, the capture nucleotide sequence is a homopolymer nucleotide sequence (e.g., a poly T sequence). As used herein, a "target nucleotide sequence" is a sequence that can hybridize with a capture nucleotide sequence (e.g., a capture nucleotide sequence present on a capture oligonucleotide). In some aspects, the target nucleotide sequence is a homopolymer nucleotide sequence (e.g., a poly A sequence).

[0077] In some aspects, the aptamer includes a protein binding nucleotide sequence, a target nucleotide sequence, and an adapter nucleotide sequence or a portion thereof. In other aspects, the aptamer includes a protein binding nucleotide sequence, a target nucleotide sequence, and an aptamer barcode nucleotide sequence. In yet another example, the present disclosure provides an aptamer complex comprising (1) an aptamer comprising (i) a capture nucleotide sequence; and (ii) an aptamer-specific nucleotide sequence; and (2) an oligonucleotide hybridized to the aptamer before the aptamer complex is contacted with a protein, the oligonucleotide comprising (i) a target nucleotide sequence; (ii) a sequence complementary to the aptamer-specific nucleotide sequence; and (iii) an aptamer barcode nucleotide sequence.

[0078] As described below, the length range of the aptamer can be about 20 to about 100 nucleotides. In some aspects, the length of the aptamer is about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides. In some aspects, the aptamer includes RNA bases. In some aspects, the aptamer includes DNA bases. In some aspects, the aptamer includes RNA and DNA bases.

[0079] method

[0080] In any aspect or aspects of the present disclosure, multiple aptamers are provided that associate with one or more proteins in a biological sample (e.g., a tissue slice), such that an aptamer, a portion of an aptamer, and / or an oligonucleotide hybridized with an aptamer is captured on a surface. In some aspects, nucleic acids (e.g., mRNA) in a biological sample (e.g., a tissue slice) are transferred to an array and captured on the array. In any aspect or aspects of the present disclosure, a co-determination is performed that provides spatial information about proteins and nucleic acids from a biological sample (e.g., a tissue slice).

[0081] For example, in some aspects, a biological sample (e.g., a tissue slice) is placed in contact with a surface, and proteins from the biological sample are associated with an aptamer. In various aspects, the aptamer is then released from the protein, and captured on the surface (e.g., by hybridization with a capture oligonucleotide) and labeled with a spatial address (barcode). In some aspects, the aptamer includes an aptamer barcode. In some aspects, the oligonucleotide hybridized with the aptamer is released after the aptamer is associated with the protein, and the oligonucleotide is captured on the surface and labeled with a spatial address (barcode). For example, the spatially labeled oligonucleotides are released and analyzed from the array by high-throughput next generation sequencing (NGS), such as synthetic sequencing (SBS). In some aspects, the capture oligonucleotide can be a universal capture oligonucleotide hybridized with, for example, an adapter region in a nucleic acid sequencing library or with a poly-A tail of an mRNA or an aptamer. In some aspects, the capture oligonucleotide can be a gene-specific capture oligonucleotide hybridized with, for example, a specifically targeted mRNA or cDNA in a sample, such as TruSeq TM Custom Amplicon (TSCA) Oligonucleotide Probes (Illumina Inc.). In some aspects, the surface includes multiple capture oligonucleotides, such as multiple identical or different capture oligonucleotides. As described herein, in any aspect or aspects of the present disclosure, the methods described herein provide for co-determination of proteins and nucleic acids (e.g., mRNA) from biological samples. See, e.g. Figure 2 .

[0082] In any aspect or aspects of the present disclosure, the method as described herein may include the step of extending the capture oligonucleotides attached to the surface, and the aptamers, oligonucleotides and / or target nucleic acids are hybridized to the capture oligonucleotides attached to the surface. In aspects where the capture oligonucleotides include barcode sequences, the resulting extended sequence will include the barcode sequence and sequences from the aptamers, oligonucleotides and / or target nucleic acids (although in complementary form). Therefore, the extended sequence is a spatially labeled version of the target nucleic acid from the biological sample.

[0083] The sequence of the extended capture oligonucleotide identifies what kind of protein and / or nucleic acid is in the biological sample, and where these proteins and / or nucleic acids are located in the biological sample. It will be understood that other sequence elements present in the capture oligonucleotide can also be included in the extension probe. Such elements include, for example, primer binding sites, cleavage sites, other tag sequences (e.g., sample identification tags), capture sequences, or combinations thereof.

[0084] Therefore, in some aspects, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at the 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; (b) allowing a plurality of aptamers to bind to the biological sample on the surface Contact, the contact causes a single aptamer in the plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer in the plurality of aptamers comprises (i) the target nucleotide sequence; (ii) an aptamer barcode nucleotide sequence; and (iii) a cleavage site; (c) removing aptamers in the plurality of aptamers that are not associated with proteins in the biological sample; (d) cleaving the plurality of aptamers to release (i) the target nucleotide sequence, and (ii) the aptamer barcode nucleotide sequence, thereby causing the target nucleotide sequence to associate with the capture nucleotide sequence, and thereby preparing the spatial proteome sequencing library. In some aspects, the surface also includes a blocker nucleic acid that hybridizes with at least a portion of the capture nucleotide sequence. In other aspects, after step (c), the blocker nucleic acid is removed from the capture oligonucleotide. In some aspects, a plurality of aptamers are cleaved by ultraviolet radiation, enzymes, or chemical cleavage. In other aspects, the method of the present disclosure also includes a step (e), i.e., extending the capture nucleotide sequence to produce a copy of a single aptamer, thereby producing an extended capture oligonucleotide. In another example, the method of the present disclosure further includes step (f), i.e., adding a template switching oligonucleotide (TSO) to the 3' end of the extended capture oligonucleotide. For example, in some aspects, a reverse transcriptase with terminal transferase activity is used, and the reverse transcriptase adds untemplated cytosine to the end of the cDNA molecule. In these aspects, a template switching oligonucleotide (TSO) having complementarity with the added cytosine is included in the reaction, thereby allowing the TSO to hybridize with the end of the cDNA molecule, and the reverse transcriptase uses it as a new template to add bases complementary to the TSO. In some examples, the method of the present disclosure also includes performing single-stranded ligation to attach a primer landing site for subsequent second-strand synthesis.

[0085] Any suitable enzymatic, chemical or photochemical cleavage reaction can be used to cleave the cleavable site. The cleavage reaction can result in a portion or all of the cleaved chain being removed. Suitable cleavage methods include, for example, restriction enzyme digestion, in which case the cleavable site is an appropriate restriction site for an enzyme that directs the cleavage of one or both chains of the duplex template; RNA enzyme digestion or chemical cleavage of the bond between deoxyribonucleotides and ribonucleotides, in which case the cleavable site can include one or more ribonucleotides; chemical reduction of disulfide bonds with a reducing agent (e.g., THPP or TCEP), in which case the cleavable site should include an appropriate disulfide bond; chemical cleavage of diol bonds with periodate, in which case the cleavable site should include diol bonds; generation of a missing base site and subsequent hydrolysis, etc. In various aspects, the cleavable site is included in the surface-immobilized primer (e.g., within the polynucleotide sequence of the primer). In various aspects, one chain of the double-stranded amplification product (or surface-immobilized primer) can include a diol bond, which allows cleavage by treatment with periodate (e.g., sodium periodate). It will be understood that more than one diol can be included at the cleavable site. One or more diol units can be incorporated into polynucleotides using the standard method of automated chemical DNA synthesis. Polynucleotide primers comprising one or more diol joints can be easily prepared by chemical synthesis. The diol joint is cracked by processing with any material (e.g., diol cracking agent) that promotes diol cracking. In all aspects, the diol cracking agent is a periodate, such as aqueous sodium periodate (NaIO4). After processing with a diol cracking agent (e.g., periodate) to crack the diol, the cracked product can be processed with a "capping agent" to neutralize the reactants produced in the cleavage reaction. Suitable capping agents for this purpose include amines, such as ethanolamine or propanolamine.

[0086] "Blocking oligonucleotide", "blocker oligonucleotide", "blocking element" or "blocker nucleic acid" refers to an agent (e.g., polynucleotide, protein, nucleotide) that reduces and / or inhibits hybridization. In various aspects, the blocker nucleic acid is a non-extendable oligomer (e.g., a 3'-blocking oligomer). The blocking element on the nucleotide can be reversible, whereby the blocking portion can be removed or modified to allow the 3' hydroxyl to form a covalent bond with the 5' phosphate of another nucleotide. For example, a reversible terminator can specify a blocking portion located at, for example, the 3' position of a nucleotide, and can be a chemically cleavable portion, such as an allyl, azidomethyl or methoxymethyl group. In various aspects, the blocking portion is irreversible (e.g., a blocking element comprising a blocking portion irreversibly prevents extension). In various aspects, the blocker nucleic acid includes an oligomer having a 3' dideoxynucleotide or similar modification to prevent polymerase extension, and is used in combination with a non-strand displacement polymerase. In another exemplary embodiment, the blocking element includes one or more modified nucleotides including a cleavable linker containing PEG (e.g., connected to a 5', 3' or nucleobase), thereby blocking extension. In another exemplary embodiment, the sealer nucleic acid includes one or more modified nucleotides, which are connected to biotin, and proteins (e.g., streptavidin) can bind to the biotin, thereby blocking polymerase extension. In another exemplary embodiment, the sealer nucleic acid includes modified nucleotides that are complementary to each other, such as iso dGTP or iso dCTP. In the polymerization reaction lacking appropriate complementary modified nucleotides, the extension of the primer is stopped. In another exemplary embodiment, the sealer nucleic acid includes one or more sequences recognized and bound by one or more single-stranded DNA binding proteins, thereby blocking the polymerase extension at the binding site. In another exemplary embodiment, the sealer nucleic acid includes one or more sequences recognized and bound by one or more short RNA or PNA oligonucleotides, thereby blocking the extension of DNA polymerases that cannot chain-displace RNA or PNA.

[0087] In another aspect, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at the 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; (b) contacting a plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer of the plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer of the plurality of aptamers comprises (i) the target nucleotide sequence; (ii) an aptamer barcode nucleotide sequence; and (iii) a truncated adapter nucleotide sequence; (c) removing aptamers of the plurality of aptamers that are not associated with proteins in the biological sample; and (d) eluting the single aptamers from the single proteins, thereby causing the target nucleotide sequence to associate with the capture nucleotide sequence, and thereby preparing the spatial proteome sequencing library. In some aspects, the surface also includes a sealant nucleic acid that hybridizes to at least a portion of the capture nucleotide sequence. In other aspects, after step (c), the sealant nucleic acid is removed from the capture oligonucleotide. In some aspects, the method of the present disclosure also includes a step (e), i.e., extending the capture nucleotide sequence to produce copies of these single aptamers, thereby producing an extended capture oligonucleotide. In another example, the method of the present disclosure also includes a step (f), i.e., hybridizing and extending the truncated adapter nucleotide sequence with a full-length adapter nucleotide sequence primer to synthesize a second chain.

[0088] In another aspect, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each capture oligonucleotide of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at the 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; (b) contacting a plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer complex of the plurality of aptamer complexes to associate with a single protein in the biological sample, wherein the plurality of aptamer complexes Each aptamer complex of comprises: (1) an aptamer, the aptamer comprising (i) the capture nucleotide sequence; and (ii) an aptamer-specific nucleotide sequence; and (2) an oligonucleotide hybridized to the aptamer prior to the contacting, the oligonucleotide comprising (i) the target nucleotide sequence; (ii) a sequence complementary to the aptamer-specific nucleotide sequence; and (iii) an aptamer barcode nucleotide sequence, wherein after the association of a single aptamer complex in the plurality of aptamer complexes with a single protein in the biological sample, the oligonucleotide is released from the aptamer, thereby causing the target nucleotide sequence of the released oligonucleotide to associate with the capture nucleotide sequence of the capture oligonucleotide in the plurality of capture oligonucleotides; thereby preparing the spatial proteome sequencing library. In some aspects, the length of the aptamer-specific nucleotide sequence is about 5 to about 20 nucleotides. In other aspects, the length of the aptamer-specific nucleotide sequence is about 10 nucleotides. In some aspects, the association of a single aptamer complex in the plurality of aptamer complexes with a single protein in the biological sample causes the oligonucleotide to be released from the aptamer. In other aspects, after the association of a single aptamer complex in the plurality of aptamer complexes with a single protein in the biological sample, conditions are changed, thereby causing release of the oligonucleotide from the aptamer. In various examples, the condition is temperature, pH, or salt concentration. In some aspects, formamide is added, thereby causing release of the oligonucleotide from the aptamer.

[0089] In any aspect or aspects of the present disclosure, the surface also includes a sealant nucleic acid that hybridizes with at least a portion of the capture nucleotide sequence. In some aspects, after the contact, the sealant nucleic acid is removed from the capture oligonucleotide. In some examples, the sealant oligonucleotide is removed from the capture oligonucleotide by exonuclease digestion. In various aspects, the exonuclease digestion is carried out using T7 exonuclease or lambda exonuclease.

[0090] In another aspect, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at the 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; (b) contacting a plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer of the plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer of the plurality of aptamers comprises (i) the target nucleotide sequence; and (ii) an aptamer barcode nucleotide sequence; (c) removing aptamers of the plurality of aptamers that are not associated with proteins in the biological sample; and (d) eluting the single aptamers from the single proteins, thereby causing the target nucleotide sequence to hybridize with the capture nucleotide sequence, and thereby preparing the spatial proteome sequencing library. In some aspects, the multiple capture oligonucleotides include a cleavable site at the 5' end. In some aspects, step (d) includes using an aptamer to competitively wash out or digest the protein in the biological sample. In some aspects, step (d) also includes contacting at least one aptamer in the multiple aptamers with a sealant nucleic acid, thereby forming a sealed aptamer, wherein the sealant nucleic acid is complementary to the target nucleotide sequence, and wherein the sealed aptamer cannot be associated with the capture nucleotide sequence. In some aspects, the surface also includes a sealant nucleic acid hybridized with at least a portion of the capture nucleotide sequence. In some aspects, after step (c), remove the sealant nucleic acid from the capture oligonucleotide. In some aspects, the method also includes (e) extending the capture nucleotide sequence to produce copies of these single aptamers, thereby producing extended capture oligonucleotides. In some aspects, step (e) further comprises hybridizing a plurality of aptamer barcoded oligonucleotides to the extended capture oligonucleotides, and extending the extended capture oligonucleotides, thereby producing a plurality of barcoded capture oligonucleotides, wherein each of the aptamer barcoded oligonucleotides comprises at least a portion of a single aptamer sequence. In some aspects, the plurality of aptamer barcoded oligonucleotides comprises a plurality of aptamer blocker nucleic acids, wherein each of the aptamer blocker nucleic acids comprises at least a portion of a single aptamer sequence.

[0091] In some aspects, 10% to 90% of the aptamers in a plurality of aptamers specific for a protein (e.g., an abundant protein) are hybridized to an aptamer blocker nucleic acid. In some aspects, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the aptamers in the plurality of aptamers specific for a protein are hybridized to an aptamer blocker nucleic acid. It will be appreciated that the amount of aptamer blocker nucleic acid included in the method can be adjusted based on the known abundance of a given target protein.

[0092] In some aspects, the method further comprises contacting a second plurality of aptamers with the biological sample on the surface, the contact causing a single aptamer in the second plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer in the second plurality of aptamers comprises a detectable portion. In some aspects, the detectable portion is a fluorophore. In some aspects, each aptamer in the second plurality of aptamers comprises the target nucleotide sequence and a truncated adapter nucleotide sequence. In some aspects, each aptamer in the second plurality of aptamers further comprises an aptamer barcode nucleotide sequence. In some aspects, after contacting the second plurality of aptamers with the biological sample on the surface, the method further comprises imaging the biological sample, thereby obtaining an image of the biological sample. In some aspects, the method does not include contacting the biological sample with a hematoxylin and eosin (H&E) staining reagent. In some aspects, at least one aptamer in the second plurality of aptamers is specific for cell membrane-associated proteins. In some aspects, at least one aptamer in the second plurality of aptamers is specific for nuclear membrane-associated proteins. In some aspects, at least one aptamer in the second plurality of aptamers is specific for a cell membrane-associated protein, and at least one aptamer in the second plurality of aptamers is specific for a nuclear membrane-associated protein, and wherein the at least one aptamer specific for the nuclear membrane-associated protein comprises a detectable portion that is different from the detectable portion comprised by the at least one aptamer specific for the cell membrane-associated protein. In some aspects, the at least one aptamer specific for the nuclear membrane-associated protein comprises an aptamer barcode nucleotide sequence that is different from the aptamer barcode nucleotide sequence comprised by the at least one aptamer specific for the cell membrane-associated protein.

[0093] In some aspects, the cell membrane-associated protein is E-cadherin, N-cadherin or Na + / K + -ATPase. Additional examples of cell membrane-associated proteins that can be targeted by the aptamers of the present disclosure include, but are not limited to, G protein-coupled receptors, epidermal growth factor receptors, P-cadherin, and R-cadherin. In some aspects, the nuclear membrane-associated protein is a nuclear pore complex protein. Examples of nuclear pore complex proteins and nuclear membrane-associated proteins that can be targeted by the aptamers of the present disclosure include, but are not limited to, Nup62, Nup107-160, Nup155, nuclear transport proteins, and Tpr.

[0094] In any aspect or aspects of the present disclosure, the biological sample is from a mammal. In various examples, the biological sample is from a human. In some aspects, the biological sample is from a mouse.

[0095] In some aspects, the disclosure also provides a method for identifying the disease in a subject suffering from the disease or at risk of suffering from the disease, the method comprising: i) according to the method disclosed herein, a spatial proteome and / or transcriptome (e.g., mRNA) library is generated from a biological sample from the subject, ii) the proteome and / or transcriptome information from the sample proteome and / or transcriptome library is compared with a control proteome and / or transcriptome library, iii) the genetic variation associated with the disease in the sample proteome and / or transcriptome library is identified. In some aspects, the disease is a neurodegenerative disease, a genetic defect or a cancer. In some aspects, the disease is Alzheimer's disease.

[0096] In any aspect or aspects of the present disclosure, the method for preparing a spatial proteome and / or transcriptome library as described herein also includes polymerase chain reaction (PCR) amplification of aptamers or other oligonucleotides captured on the surface. In other aspects, the TSO sequence is used as a primer landing site for second chain synthesis. This product is dehybridized from the surface, and PCR is performed on this product. Alternatively, the extended surface oligonucleotides can be cleaved, and PCR can be performed on this product. Sequencing adapters can be added to this product, and then placed on a second circulation pool for standard sequencing (e.g., Illumina sequencing). In any aspect or aspects of the present disclosure, PCR amplification is followed by sequencing of the amplified product to determine the identity and / or position of proteins and / or mRNA in a biological sample.

[0097] Therefore, the method described herein can be used in combination with a variety of nucleic acid sequencing techniques. In various aspects, sequencing includes sequencing by synthesis, combined sequencing, connection sequencing or pyrophosphate sequencing. In any aspect or various aspects of the present disclosure, and as described above, sequencing can occur in the second flow cell instead of the initial surface (for example, flow cell) on which the biological sample is placed. Particularly suitable technology is a technology in which nucleic acid is attached to a fixed position on the surface (for example, in an array) so that its relative position does not change and wherein the array is repeatedly imaged. The aspect of obtaining an image in different color channels (for example, matching with different marks for distinguishing a nucleotide base type from another nucleotide base type) is particularly suitable. In some aspects, the process of determining the nucleotide sequence of a target nucleic acid can be an automated process. Preferred aspects include sequencing by synthesis (" SBS ") technology. "Synthesis sequencing (" SBS ") technology " generally relates to the enzymatic extension of the nascent nucleic acid chain carried out by repeatedly adding nucleotides for the template chain. In traditional SBS methods, a single nucleotide monomer can be provided to the target nucleotide in the presence of a polymerase in each delivery. However, in the methods described herein, more than one type of nucleomonomer may be provided to a target nucleic acid in the presence of a polymerase during delivery.

[0098] In order to further illustrate the present disclosure, examples are given herein. It should be understood that these examples are provided for illustrative purposes and should not be construed as limiting the scope of the present disclosure.

[0099] Example

[0100] Example 1

[0101] Methods for spatial protein / mRNA co-determination

[0102] Provided herein are methods for determining the proteome and mRNA content of a biological sample. These methods include methods in which the proteome and mRNA content of a biological sample are co-determined. See, e.g. Figure 2 .

[0103] Figure 1 A schematic workflow for one of these methods is shown. Figure 1 A spatial proteomic workflow using an aptamer tagged with a polyA sequence at the 3' end and a truncated B15 adapter at the 5' end is shown. The truncated adapter can be used to minimize the extra bases added to the aptamer, as these extra bases may affect aptamer-protein binding. The surface capture oligonucleotide has A14 and ME sequences for downstream library preparation steps, as well as spatial barcodes, UMIs, and oligo-dT sequences to enable capture of polyA-mRNA as well as tagged aptamers.

[0104] At the beginning of the scheme, polyA oligonucleotides are optionally used to block oligo-dT sequences. Fitters are added to tissue sections and combined with their target proteins. Due to polyA closure, free fitters cannot be combined with surface oligonucleotides. After washing away free fitters, 5' to 3' exonucleases (such as T7 or λ exonucleases) are used to remove closure. Then, the labeled fitters are eluted from their targets by adding excessive unlabeled fitters, and then, the labeled fitters are diffused to the surface and captured by the surface capture oligonucleotides. Then, enzymes (e.g., reverse transcriptase or polymerase) can be used to copy the fitter sequences to the surface capture oligonucleotides. In some aspects, RNA fitters are used, and the enzyme is a reverse transcriptase. In some aspects, DNA fitters are used, and the enzyme is a polymerase. In other aspects, DNA fitters / mRNA are measured together, and the enzyme is a reverse transcriptase. Then, the truncated B15 sequence can be used as a primer to produce a complementary chain, and this complementary chain can be dehybridized from the surface. Alternatively, the capture oligonucleotide / aptamer molecules can be cleaved from the surface. PCR is then performed to add the P5 / P7 sequence and sample index to generate the final library. The aptamer sequence (which is unique for each protein target) can then be used to identify the physical location of the target protein using the spatial barcode sequence.

[0105] Figure 2 Another workflow approach is depicted. Figure 2 It is shown how the workflow of the present disclosure can be modified to be compatible with protein / mRNA co-determination. Here, a template switching reverse transcriptase (RT) is used to add a template switching oligonucleotide (TSO) sequence to the 3' end of the cDNA or copied aptamer sequence. This TSO sequence then acts as a primer landing site for copying the extended capture oligonucleotide. This second chain is dehybridized from the surface, and a solid phase reversible fixation (SPRI) step is used to separate the shorter aptamer sequence from the longer cDNA sequence. Then, the short fragment undergoes PCR to add the remaining sequencing adapters, while the long fragment is labeled, followed by PCR to add P5 / P7 and sample index. In some aspects, a ligation-based preparation is used.

[0106] Figure 3 Additional workflow approaches are presented. Figure 3The variation of the method provided herein using an aptamer including a cleavable tag is shown. Here, the aptamer includes a cleavage site, followed by a tag sequence and a polyA tail that are unique to the protein target. In this method, after the aptamer is combined with the target protein, the tag is cleaved from the aptamer and captured on the surface oligonucleotide. The cleavage can be completed using a photocleavable joint in an oligonucleotide activated by, for example, UV light, an enzymatic cleavage by USER or FPG or other targeted endonucleases, or by chemical cleavage. Then, the aptamer is copied to the surface capture oligonucleotide. This method can improve the aptamer elution and avoid aptamer replication (which may be difficult because the aptamer has a stable secondary structure).

[0107] Figure 4 An additional workflow approach is shown. Figure 4 Another variation of the method provided herein using an aptamer bound to a single-stranded DNA is shown. The aptamer undergoes conformational changes when bound to its target protein, and previous work has shown that the single-stranded DNA reporter gene bound to the aptamer can be released when the aptamer is bound to its target protein. In this method, at least partially complementary single-stranded DNA (ssDNA) molecules are bound to the aptamer to form an aptamer complex, and the aptamer complex is added to the surface before the biological sample (e.g., tissue section) is placed on the surface. In some aspects, at least partially complementary single-stranded DNA (ssDNA) molecules are bound to the aptamer to form an aptamer complex, and the aptamer complex is added to the surface after the biological sample (e.g., tissue section) is added to the surface. The binding of the aptamer to its target protein causes the release of the complementary ssDNA molecule, which is then captured on the surface by the capture oligonucleotide. This method may be beneficial because it minimizes the sequence length that needs to be added to the aptamer, and does not require specific aptamer elution conditions because the binding causes the elution of the ssDNA molecule.

[0108] Figure 5 Another workflow is depicted. Figure 5 The problem of large dynamic range and copy number of proteins is solved. Because protein levels are much higher than mRNA levels and span a wider dynamic range, it would be useful to have a mechanism to ensure that mRNA transcripts can be captured together with aptamers. To overcome this problem, the present disclosure contemplates the use of separate capture oligonucleotide sequences for mRNA and aptamer tags ( Figure 5 , top image). To address the large dynamic range of protein expression, the present disclosure contemplates using a mixture of tagged and untagged aptamers at known ratios for highly abundant proteins, such that only a small percentage of these aptamers can be captured on the surface ( Figure 5 , bottom image). Only aptamers were tagged for low-abundance proteins.

[0109] Figure 6A-6F Additional workflows are depicted. Figure 6A-6F An exemplary variation of the disclosed workflow using modified aptamers comprising unique barcode and adapter sequences is shown, with a blocker oligonucleotide complementary to the aptamer adapter sequence included to accommodate a large dynamic range of protein abundance (also referred to herein as dynamic range control). Fig. 6A is a representative illustration of the cellular environment, including a mixture of proteins (labeled "1," "2," and "X" for mRNA-binding proteins) and mRNA (depicted as a single-stranded oligonucleotide with a poly-A tail). Figure 6B The steps of permeabilizing cells (e.g., one or more cells of a tissue section) to allow modified aptamers to enter the cells are shown, wherein the aptamers target protein 1 and protein 2. The aptamers targeting each protein can include a protein-specific aptamer sequence, a barcode sequence (e.g., a barcode 1 sequence or a barcode 2 sequence), and an adapter sequence (e.g., an adapter 1 sequence or an adapter 2 sequence) from 5' to 3'. A washing step (not shown) removes unbound aptamers, such as washing with a non-ionic detergent. Examples of non-ionic detergents include, but are not limited to, Triton TM X-100, 20. 35 and 58.

[0110] Figure 6C Immobilized surface primers hybridized to blocking oligonucleotides are shown. The surface capture oligonucleotides have A14 and ME sequences for downstream library preparation steps, as well as spatial barcodes, UMIs, and oligo-dT sequences to enable capture of polyA-mRNA as well as tagged aptamers. Fig.6D The step of deblocking the surface primer to expose the free 3' end is shown. For example, the block is removed using a 5' to 3' exonuclease (such as T7 or lambda exonuclease), or by exposing the surface of the solid support to denaturing conditions (e.g., heat and / or chemical denaturants such as formamide) to dehybridize the blocker oligomer.

[0111] Fig. 6E The steps of digesting the protein to release the mRNA and aptamer, and optionally adding a dynamic range compression (DRC) blocker oligonucleotide specific for the abundant protein aptamer are shown. The protein can be digested, for example, by a protease such as proteinase K. Additional enzymes such as lipase can help facilitate retrieval of the aptamer from the permeabilized tissue / cells. Fig. 6EAs shown, the DRC blocker oligonucleotide can be complementary to one or more adapter sequences of the modified aptamer, thereby forming a blocker-aptamer complex that blocks the aptamer from hybridizing downstream with the immobilized surface primer. In some aspects, 10% to 90% of the aptamers in multiple aptamers specific for proteins (e.g., abundant proteins) are hybridized with blocker oligonucleotides. In some aspects, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% of the aptamers in the multiple aptamers specific for proteins are hybridized with blocker oligonucleotides. In some aspects, the blocker oligonucleotides include locked nucleic acids (LNA), double locked nucleic acids (bisLNA), twisted embedded nucleic acids (TINA), bridge nucleic acids (BNA), 2'-O-methyl RNA: DNA chimeric nucleic acids, minor groove binders (MGB) nucleic acids, morpholino nucleic acids, C5 modified pyrimidine nucleic acids, peptide nucleic acids (PNA), thiophosphate nucleic acids or combinations thereof.

[0112] In some aspects, the sealer nucleic acid (i.e., DRC sealer oligomer) includes one or more locked nucleic acids (LNA), 2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene functionalized oligonucleotides (TFO), pyrene functionalized oligonucleotides (PFO), peptide nucleic acids (PNA) or aminoethyl-phenoxazine-dC (AP-dC) nucleic acids. In various aspects, the sealer nucleic acid includes 10 to 15 locked nucleic acids (LNA). In various aspects, the sealer nucleic acid includes one or more thiophosphates at the 5' end. In various aspects, the sealer nucleic acid includes one or more LNAs at the 5' end. In various aspects, the sealer nucleic acid includes two or more continuous LNAs at the 3' end. In various aspects, the sealer nucleic acid includes two or more continuous LNAs at the 5' end. In various aspects, the sealer nucleic acid includes a plurality of (e.g., 2 to 10) synthetic nucleotides (e.g., LNA) and a plurality of (e.g., 2 to 10) canonical or natural nucleotides (e.g., dNTPs).

[0113] Fig. 6F Shown is the step of capturing the released mRNA and aptamer barcode on an immobilized surface primer, followed by RT-PCR / PCR to produce covalently attached complementary sequences of mRNA and aptamer barcodes. The aptamer hybridized with the DRC blocker oligonucleotide is not captured by the surface primer and can be washed away. Then, PCR is performed to add the P5 / P7 sequence and sample index to produce the final library. Then, the aptamer sequence (which is unique for each protein target) can be used to identify the physical location of the target protein using a spatial barcode sequence. The difference in the barcode or sequence itself present in the mRNA capture primer allows the mRNA sequence to be distinguished from the aptamer sequence.

[0114] Figure 7A-7DAlternative workflows are depicted. Figure 7A-7D The use of DRC blocking agents to maintain abundant aptamers in solution (e.g., SomaLogic (SomaLogic)) rather than being captured on the surface of a solid support. The tissue section is permeabilized to allow the aptamer to enter the cell without losing mRNA. After adding the aptamer, unbound aptamer is washed away, for example using a non-ionic detergent. Examples of non-ionic detergents include, but are not limited to, Triton TM X-100, 20. 35 and 58. Cellular proteins are then digested, for example with proteinase K, thereby releasing the protein-bound aptamer and any protein-bound mRNA. The immobilized capture oligonucleotide on the solid support is then unblocked, thereby exposing the free 3' end, thereby allowing hybridization with the mRNA and aptamer sequences.

[0115] like Fig. 7A As shown, after cell permeabilization, aptamer binding and protein digestion, the released mRNA and SOMA polymers are captured and copied to the immobilized surface primer. STUBBY corresponds to the universal adapter sequence at the 3' end of the aptamer (i.e., SOMA polymer). DRC blocking agent (i.e., a blocking agent oligomer including the complementary sequence of the STUBBY sequence and the complementary sequence of the SOMA polymer sequence) is then added to the SOMA polymer. Figure 7B The following steps are shown: After replication, unbound mRNA and SOMA-mers are washed away, leaving behind an immobilized, extended surface primer comprising either the cDNA of the mRNA or the complementary sequence of the STUBBY and SOMA-mer sequences. Figure 7C The steps of adding a SOMA polymer barcode to an extended surface primer including a complementary sequence to a SOMA polymer sequence are shown, wherein an oligomer including a SOMA polymer-SEQ sequence, a barcode sequence, and a B-15' adapter sequence is hybridized to the extended surface primer and copied. This oligomer can include a B-15' adapter sequence, a barcode sequence, and a sequence complementary to the SOMA polymer sequence (e.g., SOMA polymer-SEQ) from 5' to 3'. A "dummy" oligomer can also be included, which lacks a barcode sequence and a B-15' sequence and can act as a DRC blocker to prevent replication and sequencing of highly abundant SOMA polymers.

[0116] Fig.7DA cleavage step is shown to release the extended surface primers including the SOMAmer barcode and the B-15 adapter sequence, followed by a library preparation step. Cleavage can be accomplished using a photocleavable linker in the oligonucleotide activated by, for example, UV light, enzymatic cleavage by USER or FPG or other targeted endonucleases, or by chemical cleavage.

[0117] Example 2

[0118] Methods for spatial targeting of cell membranes

[0119] Provided herein are methods for measuring biological samples (e.g., cell membranes and nuclear membranes) using fluorescently labeled aptamers. These methods include methods in which the proteome and mRNA content of a biological sample are co-measured.

[0120] Figure 8A-8B A schematic workflow for one of these methods is shown. Figure 8A-B An exemplary variation of the disclosed workflow using fluorescently labeled aptamers targeted to the cell membrane is shown, for example for cell segmentation analysis and image alignment processing. Fig. 8A The following steps are shown: adding a fluorescently labeled aptamer to the protein, allowing any unbound aptamer to flow away, and then imaging the tissue to visualize the bound aptamer. For example, a fluorescence microscope can be used to image the bound fluorescently labeled aptamer. The labeled aptamer includes a poly-A sequence at the 3' end (e.g., adjacent to the fluorophore) and a truncated B15 adapter at the 5' end. The truncated adapter can be used to minimize the extra bases added to the aptamer, as these extra bases may affect aptamer-protein binding. The surface capture oligonucleotide has A14 and ME sequences for downstream library preparation steps, as well as spatial barcodes, UMIs, and oligo-dT sequences to enable capture of polyA-mRNA as well as tagged aptamers.

[0121] The immobilized surface primer on the solid support is blocked with, for example, a complementary blocking oligonucleotide. At the beginning of the protocol, a polyA oligonucleotide is optionally used to block the oligo-dT sequence. The aptamer is added to the tissue section and binds to its target protein. Due to the polyA blocking, the free aptamer cannot bind to the surface oligonucleotide. Figure 8BShown is a step of removing blocking oligomers from surface primers using, for example, T7 or lambda exonucleases. Then, the aptamer labeled is eluted from its target by adding an excessive amount of unlabeled aptamer, and then, the aptamer labeled is diffused to the surface and captured by the surface capture oligonucleotide. Then, an enzyme (e.g., reverse transcriptase or polymerase) can be used to copy the aptamer sequence to the surface capture oligonucleotide. In some aspects, RNA aptamers are used, and the enzyme is a reverse transcriptase. In some aspects, DNA aptamers are used, and the enzyme is a polymerase. In other aspects, DNA aptamers / mRNA are measured together, and the enzyme is a reverse transcriptase. Then, the truncated B15 sequence can be used as a primer to produce a complementary chain, and this complementary chain can be dehybridized from the surface. Alternatively, oligonucleotide / aptamer molecules can be captured from surface cleavage. Then, PCR is performed to add P5 / P7 sequences and sample indexes to produce a final library. Then, the aptamer sequence (which is unique for each protein target) can be used to identify the physical location of the target protein using a spatial barcode sequence.

[0122] Figure 9A-9B Various examples of cell membrane-targeting aptamers and nuclear membrane-targeting aptamers are shown. Fig. 9A Various types of cell membrane targeting aptamers are shown that include a common fluorophore and barcode to, for example, enable membrane detection across different tissue types. Each aptamer can include a cell membrane barcode and a truncated B15 adaptor sequence. Fig. 9B Exemplary aptamers for co-detection of cell membrane and nuclear membrane proteins using differently labeled aptamers (e.g., aptamers with different fluorophores and barcode sequences) are shown. In some aspects, the cell membrane-associated protein is E-cadherin, N-cadherin, or Na + / K + -ATPase. In some aspects, the nuclear membrane protein is a nuclear pore complex protein.

Claims

1. A method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at a 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence and a unique molecular identifier sequence shared between oligonucleotides within a spatial feature; (b) contacting a plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer of the plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer in the plurality of aptamers comprises (i) the target nucleotide sequence; (ii) an aptamer barcode nucleotide sequence; and (iii) a cleavage site; (c) removing the aptamers that are not associated with the protein in the biological sample from the plurality of aptamers; (d) cleaving the plurality of aptamers to release (i) the target nucleotide sequence, and (ii) the aptamer barcode nucleotide sequence, thereby allowing the target nucleotide sequence to associate with the capture nucleotide sequence, and Thus, the spatial proteome sequencing library is prepared.

2. The method of claim 1, wherein the surface further comprises a blocking agent nucleic acid that hybridizes to at least a portion of the capture nucleotide sequence. 3 . The method of claim 2 , wherein after step (c), the blocker nucleic acid is removed from the capture oligonucleotide.

4. The method according to any one of claims 1 to 3, wherein the plurality of aptamers are cleaved by ultraviolet radiation, enzymatic or chemical cleavage.

5. The method of any one of claims 1 to 4, further comprising (e) extending the capture nucleotide sequence to generate copies of the single aptamer, thereby generating an extended capture oligonucleotide. 6 . The method of claim 5 , further comprising (f) adding a template switching oligonucleotide (TSO) to the 3′ end of the extended capture oligonucleotide. The method of claim 6 , wherein the TSO is directly linked to the extended capture oligonucleotide.

8. A method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each capture oligonucleotide of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at the 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence and a unique molecular identifier sequence shared between oligonucleotides within the spatial feature; (b) contacting a plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer of the plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer of the plurality of aptamers comprises (i) the target nucleotide sequence; (ii) an aptamer barcode nucleotide sequence; and (iii) a truncated adapter nucleotide sequence; (c) removing the aptamers that are not associated with the protein in the biological sample from the plurality of aptamers; (d) eluting the single aptamer from the single protein, thereby allowing the target nucleotide sequence to associate with the capture nucleotide sequence, and Thus, the spatial proteome sequencing library is prepared.

9. The method of claim 8, wherein the surface further comprises a blocker nucleic acid that hybridizes to at least a portion of the capture nucleotide sequence.

10. The method of claim 9, wherein after step (c), the blocker nucleic acid is removed from the capture oligonucleotide.

11. The method of any one of claims 8 to 10, further comprising (e) extending the capture nucleotide sequence to generate copies of the single aptamer, thereby generating an extended capture oligonucleotide. 12 . The method according to claim 11 , further comprising (f) hybridizing and extending the truncated adapter nucleotide sequence with a full-length adapter nucleotide sequence primer to synthesize a second strand.

13. A method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each capture oligonucleotide of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at the 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence and a unique molecular identifier sequence shared between oligonucleotides within the spatial feature; (b) contacting a plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer complex of the plurality of aptamer complexes to associate with a single protein in the biological sample, wherein each aptamer complex of the plurality of aptamer complexes comprises: (1) an aptamer, the aptamer comprising (i) the capture nucleotide sequence; and (ii) an aptamer-specific nucleotide sequence; and (2) an oligonucleotide hybridized to the aptamer before the contacting, the oligonucleotide comprising (i) the target nucleotide sequence; (ii) a sequence complementary to the aptamer-specific nucleotide sequence; and (iii) an aptamer barcode nucleotide sequence, wherein following said association of a single aptamer complex among said plurality of aptamer complexes with a single protein in said biological sample, said oligonucleotide is released from said aptamer, thereby allowing said target nucleotide sequence of said released oligonucleotide to associate with said capture nucleotide sequence of a capture oligonucleotide among said plurality of capture oligonucleotides; Thus, the spatial proteome sequencing library is prepared.

14. The method of claim 13, wherein the aptamer-specific nucleotide sequence is about 5 to about 20 nucleotides in length.

15. The method of claim 13, wherein the aptamer-specific nucleotide sequence is about 10 nucleotides in length.

16. The method of any one of claims 1 to 15, wherein the surface further comprises a blocker nucleic acid that hybridizes to at least a portion of the capture nucleotide sequence. The method of claim 16 , wherein following the contacting, the blocker nucleic acid is removed from the capture oligonucleotide.

18. The method of any one of claims 13 to 17, wherein the association of a single aptamer complex of the plurality of aptamer complexes with a single protein in the biological sample results in the release of the oligonucleotide from the aptamer.

19. The method according to any one of claims 13 to 17, wherein after the association of a single aptamer complex among the plurality of aptamer complexes with a single protein in the biological sample, conditions are changed, thereby causing release of the oligonucleotide from the aptamer.

20. The method of claim 19, wherein the condition is temperature, pH or salt concentration.

21. The method according to any one of claims 13 to 20, wherein after the association of a single aptamer complex among the plurality of aptamer complexes with a single protein in the biological sample, formamide is added, thereby releasing the oligonucleotide from the aptamer.

22. The method of any one of claims 3, 10 or 17, wherein the blocking agent oligonucleotide is removed from the capture oligonucleotide by exonuclease digestion.

23. The method of claim 22, wherein the exonuclease digestion is performed using T7 exonuclease or lambda exonuclease.

24. A method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface, the surface comprising: a plurality of capture oligonucleotides immobilized on the surface, wherein each capture oligonucleotide of the plurality of capture oligonucleotides comprises (i) a capture nucleotide sequence at the 3' end, the capture nucleotide sequence being configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence and a unique molecular identifier sequence shared between oligonucleotides within the spatial feature; (b) contacting a plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer of the plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer in the plurality of aptamers comprises (i) the target nucleotide sequence; and (ii) an aptamer barcode nucleotide sequence; (c) removing the aptamers that are not associated with the protein in the biological sample from the plurality of aptamers; (d) digesting the protein in the biological sample, thereby releasing the single aptamer from the single protein, thereby allowing the target nucleotide sequence to hybridize with the capture nucleotide sequence, and thereby preparing the spatial proteome sequencing library.

25. The method of claim 24, wherein the plurality of capture oligonucleotides comprises a cleavable site at the 5' end.

26. The method of claim 24 or 25, wherein step (d) further comprises contacting at least one of the plurality of aptamers with a blocking agent nucleic acid, thereby forming a blocked aptamer, wherein the blocking agent nucleic acid is complementary to the target nucleotide sequence, and wherein the blocked aptamer is unable to associate with the capture nucleotide sequence.

27. The method of any one of claims 24 to 26, wherein the surface further comprises a blocker nucleic acid that hybridizes to at least a portion of the capture nucleotide sequence.

28. The method of claim 27, wherein after step (c), the blocker nucleic acid is removed from the capture oligonucleotide.

29. The method of any one of claims 24 to 28, further comprising (e) extending the capture nucleotide sequence to generate copies of the single aptamer, thereby generating an extended capture oligonucleotide.

30. The method of claim 29, wherein step (e) further comprises hybridizing a plurality of aptamer barcoded oligonucleotides to the extended capture oligonucleotide and extending the extended capture oligonucleotide, thereby producing a plurality of barcoded capture oligonucleotides, wherein each of the aptamer barcoded oligonucleotides comprises at least a portion of a single aptamer sequence.

31. The method of claim 30, wherein the plurality of aptamer barcoding oligonucleotides comprises a plurality of aptamer blocker nucleic acids, wherein each of the aptamer blocker nucleic acids comprises at least a portion of a single aptamer sequence.

32. The method of any one of claims 24 to 31, further comprising cleaving the cleavable site, thereby releasing the plurality of capture oligonucleotides from the surface.

33. The method of any one of claims 1 to 32, wherein the aptamer comprises a detectable moiety.

34. The method of claim 33, wherein the detectable moiety is a fluorophore.

35. The method of any one of claims 1 to 34, wherein the method further comprises contacting a second plurality of aptamers with the biological sample on the surface, the contacting causing a single aptamer in the second plurality of aptamers to associate with a single protein in the biological sample, wherein each aptamer in the second plurality of aptamers comprises a detectable portion.

36. The method of claim 35, wherein the detectable moiety is a fluorophore.

37. The method of claim 35 or 36, wherein each aptamer in the second plurality of aptamers comprises the target nucleotide sequence and a truncated adapter nucleotide sequence.

38. The method of claim 37, wherein each aptamer in the second plurality of aptamers further comprises an aptamer barcode nucleotide sequence.

39. The method of any one of claims 35 to 38, wherein after contacting the second plurality of aptamers with the biological sample on the surface, the method further comprises imaging the biological sample, thereby obtaining an image of the biological sample.

40. The method of claim 39, wherein the method does not comprise contacting the biological sample with a hematoxylin and eosin (H&E) staining reagent.

41. The method of any one of claims 35 to 40, wherein at least one aptamer in the second plurality of aptamers is specific for a cell membrane associated protein.

42. The method of any one of claims 35 to 40, wherein at least one aptamer in the second plurality of aptamers is specific for a nuclear membrane associated protein.

43. The method of any one of claims 35 to 40, wherein at least one aptamer in the second plurality of aptamers is specific for a cell membrane-associated protein, and at least one aptamer in the second plurality of aptamers is specific for a nuclear membrane-associated protein, and wherein the at least one aptamer specific for the nuclear membrane-associated protein comprises a detectable portion that is different from the detectable portion comprised by the at least one aptamer specific for the cell membrane-associated protein.

44. The method according to any one of claims 38 to 43, wherein the at least one aptamer specific for the nuclear membrane-associated protein comprises an aptamer barcode nucleotide sequence that is different from the aptamer barcode nucleotide sequence comprised by the at least one aptamer specific for the cell membrane-associated protein.

45. according to the method described in any one in claim 41 to 44, wherein said cell membrane associated protein is E-cadherin, N-cadherin or Na + / K + -ATPase.

46. ​​The method of any one of claims 42 to 44, wherein the nuclear membrane associated protein is a nuclear pore complex protein.

47. The method of any one of claims 1 to 46, wherein the biological sample is from a mammal.

48. The method of any one of claims 1 to 47, wherein the biological sample is from a human.

49. A method of identifying a disorder in a subject having or at risk of having a disorder, the method comprising: i) generating a spatial proteomic and / or transcriptomic library from a biological sample from said subject according to the methods of the present disclosure, ii) comparing the proteome and / or genetic information from the sample proteome and / or transcriptome library with a control proteome and / or transcriptome library, iii) identifying genetic variations associated with the disease in the sample proteome and / or transcriptome library.

50. The method of claim 49, wherein the disorder is a neurodegenerative disorder.

51. The method of claim 49 or 50, wherein the disorder is Alzheimer's disease.

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